Administration of interferons of both the γ and alfa/β classes down-regulates hepatic cytochrome P450 (CYP) genes when administered to humans or rats. In male rats, interferons decrease expression of CYP3A2 at a pretranslational level, but because interferons also release other cytokines in vivo, it is unclear whether this is a direct effect on hepatocytes. We therefore examined the effects of rat recombinant interferon γ (IFN-γ) on CYP3A2, other 3A genes, and 2C11 in stable primary cultures of male rat hepatocytes. Hepatocytes were cultured on matrigel in Williams' E, and messenger RNAs (mRNAs) for 3A2, 3A1-like CYPs, and 2C11 mRNA were determined by RNase protection assays. CYP3A and 2C11 proteins were immunoquantified, and their catalytic activities were estimated by testosterone hydroxylation pathways. In control cells, 3A2 mRNA decreased initially but then recovered, and stable levels (15% of freshly isolated cells) were attained between days 3 and 7. Phenobarbital increased 3A2 mRNA to 60–120% values of freshly isolated cells, and mRNA for 3A1-like CYPs were increased 20-fold. In both control and phenobarbital-treated hepatocytes, rat recombinant IFN-γ (33 U/mL) reduced mRNA for 3A2 and 3A1-like CYPs, as well as 3A protein and testosterone 6 β-hydroxylase activity. Interferon had no effect on CYP2C11 at mRNA or protein levels in untreated cells, although a reduction in 2C11 protein was evident in phenobarbital-treated cultures. It is concluded that interferon directly alters expression of constitutive and inducible CYP3A genes in well-differentiated male rat hepatocytes in culture, but has no effect on constitutive expression of CYP2C11.