We have used maximum-likelihood models of codon substitution to investigate the role of adaptive evolution in the evolution of cytochrome P450 (CYP) sequences. Evidence for the operation of adaptive evolution in the evolution of rat CYP2C, rabbit CYP2C, rat CYP2D, human CYP3A and rabbit CYP4A was observed. The absence of signal in rat CYP2B, rat CYP3A, human CYP2C and monkey CYP2C suggests that the adaptive evolution did not operate in the evolution of these cytochromes. Our results show identical adaptive evolution patterns for rabbit (lagomorpha) and rat (rodentia) CYP2C. The absence of signal for adaptive evolution in primate CYP2C suggests that the identical rat and rabbit CYP2C patterns arose in the last common ancestor of rodentia and lagomorpha. Furthermore, we have found statistically significant association of sites under adaptive evolution and Gotoh's substrate recognition sites in rat and rabbit CYP2C (5%), human CYP3A and rat CYP2D (10%). From these correlations, the given substrate-dependent nature of differences in CYP substrate-specificity profiles and differences in cytochrome active-site residues, we hypothesize that the most likely role of adaptive evolution in the evolution of cytochrome P450 substrate specificities was to fix mutations that permitted an increased number of binding modes (thereby expanding the substrate repertoire). The pattern of adaptive evolution observed in this work is consistent with results from microsomal studies in which CYP2C isoforms are responsible for most of the metabolism of foreign compounds in rat and rabbit, and CYP3A isoforms play the same role in humans.