The kinetics of CO binding to cytochromes P450, measured by the flash photolysis technique, were used to probe the interaction of erythromycin with cytochromes P450 in rat liver microsomes. Addition of erythromycin generates substrate difference spectra using microsomes from rats treated with phenobarbital or dexamethasone but not from untreated rats, showing that it binds to P450s induced by these agents. In contrast, erythromycin and/or a monoclonal antibody to P450 3A1/2 accelerated CO binding to microsomes from rats treated with phenobarbital but had no effect on microsomes from untreated or dexamethasone-treated rats. Based on the differential amounts and inducibilities of the P450 3A1 and 3A2 forms in these microsomal samples, these results indicate that erythromycin increased the rate for P450 3A2 but not P450 3A1. The divergent effects of erythromycin on these P450s, which exhibit 89% sequence similarity, were consistent with a model of the P450 substrate binding site in which erythromycin forms a more rigid complex with P450 3A1 than P450 3A2. These results demonstrate the sensitivity of P450 conformation/dynamics to substrate binding, and show that CO binding kinetics can distinguish among closely related P450s in a microsomal environment.