We previously reported that in vivo cimetidine inhibits hepatic microsomal enzyme activities mediated by cytochrome P450 (CYP)2C11 and at least one other CYP enzyme but does not inhibit CYP2A1-, CYP2B- or CYP3A-mediated activities in adult male rats. To investigate the effects of in vivo cimetidine on CYP1A1, cimetidine (150 mg/kg i.p.) or saline was administered to beta-naphthoflavone-induced (40 mg/kg i.p. once daily for 3 consecutive days) or uninduced adult male Wistar rats, and hepatic microsomes were prepared 90 min after the cimetidine injection. Cimetidine had no effect on either methoxyresorufin O-dealkylase (MROD) or ethoxyresorufin O-dealkylase (EROD) activity in microsomes from beta-naphthoflavone-induced rats. In these same microsomes, polyclonal anti-CYP1A1 IgG inhibited both MROD and EROD activities by >90%, whereas monoclonal anti-CYP1A1 IgG inhibited MROD and EROD activities by 60% and 80%, respectively. In contrast, cimetidine inhibited MROD and EROD activities in microsomes from uninduced rats by 50% and 65%, respectively (P < .05). Immunoinhibition studies with polyspecific and monospecific anti-CYP2C11 IgG indicated that MROD and EROD activities are mediated by a CYP2C enzyme or enzymes other than CYP2C11 in these microsomes. To investigate the possibility that the drug affected EROD activity in uninduced rats by inhibiting CYP2C6, cimetidine was administered as described to rats that had been pretreated with phenobarbital (80 mg/kg i.p once daily for 4 consecutive days). In hepatic microsomes from these rats, cimetidine inhibited progesterone 21-hydroxylase activity (mediated by CYP2C6) by 62% and progesterone 2alpha-hydroxylase activity (mediated by CYP2C11) by 39% but had no effect on progesterone 6beta-hydroxylase activity (mediated by CYP3A). Taken together, the results indicate that in vivo cimetidine has no effect on CYP1A1 but inhibits CYP2C6 in addition to CYP2C11. Preincubation of microsomes from uninduced rats with cimetidine and NADPH in vitro increased the potency of inhibition of EROD activity by 20-fold, suggesting that cimetidine inhibits CYP2C6, as it does CYP2C11: by forming a metabolite/intermediate complex.