The way in which structural diversity encodes the capacity of individual P450 enzymes to metabolize multiple, structurally distinct substrates remains largely unknown. The tools of molecular biology provide a means of identifying amino acid residues among closely related P450s that are determinants of their distinct catalytic properties. Work in our laboratory has identified two substrate specificity‐determining segments of the amino acid sequences of subfamily 2C P450s. A pattern has emerged from this work, and that of others, which suggests a model for the structural basis of P450 catalytic diversity.—Johnson, E. F.; Kronbach, T.; Hsu, M.‐H. Analysis of the catalytic specificity of cytochrome P450 enzymes through site‐directed mutagenesis.FASEB J.6: 700‐705; 1992.