Eleuthère Irénée du Pont (1771-1834), founder of E. I. du Pont de Nemours & Co., and Sophie Madeleine Dalmas du Pont (1775-1828) had three sons: Alfred Victor du Pont (1798-1856), Henry du Pont (1812-1889), and Alexis I. du Pont (1816-1857). All three were partners in their father's company.

Alfred Victor du Pont was born in Paris and came to the United States in 1800. He was educated at Mt. Airy and Dickinson colleges in Pennsylvania. He studied chemistry at Dickinson under Thomas Cooper, a disciple of Joseph Priestly. In 1818, he joined the firm E. I. du Pont de Nemours & Co. Upon the death of his father in 1834, he formed a new partnership with his brother-in-law, James Antoine Bidermann. In 1837, Alfred Victor du Pont became the senior partner. He retired in 1850. He was primarily interested in developing methods for improving the manufacture of black powder. He worked with Ellwood Morris, a Philadelphia engineer, evaluating the efficiency of turbines at the Du Pont mills.

In 1824, Alfred V. du Pont married Margaretta Elizabeth Lammot (1807-1898), daughter of Daniel Lammot (1782-1877) and Susan Parham Beck (1786-1817). They resided at Nemours, a house built for them in 1824 near the home of his father. He was active in the local school and political affairs, and served as a Presidential Elector in 1844. Margaretta Lammot du Pont and her father were active in establishing the Wilmington congregation of Swedenborgians (Church of the New Jerusalem) in Delaware. Of Alfred V. du Pont's seven children, two sons, Eleuthère Irénée du Pont (1829-1877) and Lammot du Pont (1831-1884), were active in the firm of E. I. du Pont de Nemours & Co. After her husband's death, Margaretta Lammot du Pont moved to another residence, Goodstay, located in Wilmington, where she resided until her death.

Henry du Pont was an American military officer. He was born in 1812 at Eleutherian Mills near Wilmington, Delaware. He was educated at Mt. Airy College, Germantown, Pennsylvania, and at the United States Military Academy at West Point from 1829 to 1833. Upon his graduation from West Point in 1833, he was commissioned a brevet second lieutenant in the Fourth Artillery. He served at Fort Monroe, Virginia, and at Fort Mitchell, Alabama, when he resigned his commission at the request of his father and returned to Delaware to manage E. I. du Pont de Nemours & Co. In 1837, he and his brothers became partners in the firm. In 1850, he became the senior partner and director, continuing in this capacity until his death in 1889. Under his direction, the company purchased mills on Big Wapwallopen Creek in Luzerne County, Pennsylvania, in 1859 in order to supply blasting powder for the nearby Wyoming and Hazleton coal fields. This was the company's first operation outside Delaware. In 1872, Henry du Pont, with his nephew Lammot, took a leading part in the formation of the Gunpowder Trade Association to control the powder companies that had sprung up during the Civil War. Under his leadership, Du Pont first became a national organization.

In 1837, Henry du Pont married Louisa Gerhard (1816-1900). She was the daughter of William Gerhard (1774-1848) and Sarah Wood Gerhard (1781-1851). The couple had nine children and resided in the Du Pont family home, Eleutherian Mills, located north of Wilmington, Delaware.

In 1841, Henry du Pont was on the staff of the Governor of Delaware, and from 1846 to 1861 was Adjutant-General for the State of Delaware. In 1861, he was made Major General of the state troops, commanding Delaware's volunteers; thereafter, he was known as "The General." He is credited with doing much to keep Delaware on the Union side during the Civil War.

Henry du Pont was active in Delaware politics, first as a Whig and then as a Republican. He also farmed and became the third owner of Winterthur, one of his family's estates.

Alexis I. du Pont was the youngest child. He was educated at Mt. Airy College in Germantown, Pennsylvania, and at the New Haven Gymnasium from 1829 to 1831, where his nephew, James Eleuthère Bidermann (1790-1865), was a classmate. In 1831, he entered the University of Pennsylvania, where he completed his scientific studies in 1835 but did not earn a degree.

In 1836, he married Joanna Maria Smith (1815-1876), the daughter of Francis Gurney Smith (1784–1873) of Philadelphia and a sister of Dr. Thomas Mackie Smith (1809-1852), who had married Alexis I. du Pont's sister Eleuthera du Pont Smith (1806-1876). She attended Miss Ardley's French School in Philadelphia during the late 1820s. Alexis I. du Pont and Joanna Maria Smith du Pont had eight children.

Alexis I. du Pont became a partner in E. I. du Pont de Nemours & Company in 1837 and continued as such until his death on August 23, 1857, having been fatally injured in an explosion at the powder mills on the preceding day.