Joining the faculty of Kazan University in 1849, Butlerov took up the new theories of the French chemists Auguste Laurent and Charles Gerhardt and worked on new methods of synthesizing known compounds and on entirely new synthetics.

In 1861 Butlerov stated his concept of chemical structure: that the chemical nature of a molecule is determined not only by the number and type of atoms but also by their arrangement. He foresaw and demonstrated the existence of isomers (molecules composed of the same atoms but in different arrangements), such as the two butanes and three pentanes. In 1866 he synthesized isobutane. Two years later he discovered that unsaturated organic compounds contain multiple bonds.