The substrate recognition regions in cytochrome P450 family 2 (CYP2) proteins were inferred by group-to-group alignment of CYP2 sequences and those of bacterial P450s, including Pseudomonas putida P450 101A (P450cam), whose substrate-binding residues have been definitely identified by x-ray crystallography of a substrate-bound form (Poulos T. L., Finzel, B. C., and Howard, A. J. (1987) J. Mol. Biol. 195, 687-700). The six putative substrate recognition sites, SRSs, thus identified are dispersively located along the primary structure and constitute about 16% of the total residues. All the reported point mutations and chimeric fragments that significantly affect the substrate specificities of the parental CYP2 enzymes fell within or overlapped some of the six SRSs. Analysis of nucleotide substitution patterns in closely related members in four subfamilies, CYP2A, 2B, 2C, and 2D, consistently indicated that the SRSs have accumulated more nonsynonymous (amino acid-changing) substitutions than the rest of the sequence. This observation supports the idea that diversification of duplicate genes of drug-metabolizing P450s occurs primarily in substrate recognition regions to cope with an increasing number of foreign compounds.