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With the aid of a grant of money from the king of Prussia, Agassiz crossed the Atlantic in the autumn of 1846 to investigate the natural history and geology of North America and to deliver a course of lectures on "The Plan of Creation as shown in the Animal Kingdom" by invitation from John Amory Lowell, at the Lowell Institute in Boston, Massachusetts. The financial offers that were presented to him in the United States induced him to settle there, where he remained to the end of his life. He was elected a foreign honorary member of the American Academy of Arts and Sciences in 1846.

In 1846, still married to Cecilie, who remained with their three children in Switzerland, Agassiz met Elizabeth Cabot Cary at a dinner. The two developed a romantic attachment, and when his wife died in 1848, they made plans to marry. The ceremony took place on April 25, 1850, in Boston, Massachusetts at King's Chapel. Agassiz brought his children to live with them, and Elizabeth raised and developed close relationships with her step-children. She had no children of her own.

Agassiz had a mostly cordial relationship with the Harvard botanist Asa Gray despite their disagreements. Agassiz believed each human race had been separately created, but Gray, a supporter of Charles Darwin, believed in the shared evolutionary ancestry of all humans. In addition, Agassiz was a member of the Scientific Lazzaroni, a group of mostly physical scientists who wanted American academia to mimic the more autocratic academic structures of European universities, but Gray was a staunch opponent of that group.

Agassiz's engagement for the Lowell Institute lectures precipitated the establishment in 1847 of the Lawrence Scientific School at Harvard University, with Agassiz as its head. Harvard appointed him professor of zoology and geology, and he founded the Museum of Comparative Zoology there in 1859 and served as its first director until his death in 1873. During his tenure at Harvard, Agassiz studied the effect of the last ice age in North America. In August 1857, Agassiz was offered the chair of palaeontology in the Museum of Natural History, Paris, which he refused. He was later decorated with the Cross of the Legion of Honor.

Agassiz continued his lectures for the Lowell Institute. In succeeding years, he gave lectures on "Ichthyology" (1847–1848), "Comparative Embryology" (1848–1849), "Functions of Life in Lower Animals" (1850–1851), "Natural History" (1853–1854), "Methods of Study in Natural History" (1861–1862), "Glaciers and the Ice Period" (1864–1865), "Brazil" (1866–1867), and "Deep Sea Dredging " (1869–1870). In 1850, he had married Elizabeth Cabot Cary, who later wrote introductory books about natural history and a lengthy biography of her husband after he had died.

Agassiz served as a nonresident lecturer at Cornell University while he was also on faculty at Harvard. In 1852, he accepted a medical professorship of comparative anatomy at Charlestown, Massachusetts, but he resigned in two years. From then on, Agassiz's scientific studies dropped off, but he became one of the best-known scientists in the world. By 1857, Agassiz was so well-loved that his friend Henry Wadsworth Longfellow wrote "The Fiftieth Birthday of Agassiz" in his honor and read it at a dinner given for Agassiz by the Saturday Club in Cambridge. Agassiz's own writing continued with four (of a planned 10) volumes of Natural History of the United States, published from 1857 to 1862. He also published a catalog of papers in his field, Bibliographia Zoologiae et Geologiae, in four volumes between 1848 and 1854.

Stricken by ill health in the 1860s, Agassiz resolved to return to the field for relaxation and to resume his studies of Brazilian fish. In April 1865, he led the Thayer Expedition to Brazil. While there, he commissioned two photographers, Augusto Stahl and Georges Leuzinger, to accompany the expedition and produce somatological images of Indigenous people and enslaved Africans and Black people. After his return in August 1866, an account of the expedition, A Journey in Brazil, was published in 1868. In December 1871, he made a second eight-month excursion, known as the Hassler expedition under the command of Commander Philip Carrigan Johnson (the brother of Eastman Johnson) and visited South America on its southern Atlantic and Pacific Seaboards. The ship explored the Magellan Strait, which drew the praise of Charles Darwin.

Following the establishment of the first U.S. Society for the Prevention of Cruelty to Animals in New York City in 1866, Agassiz was called on to help settle disputes about animal behavior. He deemed the way turtles were shipped caused them suffering, while P.T. Barnum argued with Agassiz' support that his snakes would eat only live animals.

His second wife, Elizabeth Cary Agassiz, assisted him in preparing his A Journey in Brazil. Along with her stepson, Alexander Agassiz, she wrote Seaside Studies in Natural History and Marine Animals of Massachusetts. Elizabeth wrote at the Strait that "the Hassler pursued her course, past a seemingly endless panorama of mountains and forests rising into the pale regions of snow and ice, where lay glaciers in which every rift and crevasse, as well as the many cascades flowing down to join the waters beneath, could be counted as she steamed by them.... These were weeks of exquisite delight to Agassiz. The vessel often skirted the shore so closely that its geology could be studied from the deck."