Three recombinant human leukocyte interferons (IFLrA, IFLrD, and a hybrid IFLrA/D) that differ markedly in their antiviral activity in murine L cells were examined for their effects on hepatic microsomal drug metabolism in adult female CD-1 mice. When administered for 1 or 3 consecutive days, IFLrA/D, which exhibited the highest antiviral activity in murine L cells, caused a dose-dependent decrease in cytochrome P-450 content and in the rate of metabolism in vitro of benzo[a]pyrene, hexobarbital, 7-ethoxycoumarin, benzphetamine, and zoxazolamine. The concentration of cytochrome b and the activity of NADPH-cytochrome c reductase were also depressed when IFLrA/D was administered for 3 days. Similar but somewhat smaller changes were observed following treatment of mice with IFLrD, which possessed approximately 1% of the antiviral activity of IFLrA/D in murine L cells. In contrast, IFLrA, which was essentially devoid of antiviral activity in the mouse cell line, failed to depress cytochrome P-450 levels and in vitro drug metabolism activity in a consistent or dose-dependent manner. Cytochrome P-450 content and the in vitro rate of metabolism of benzphetamine and zoxazolamine were maximally depressed 8-24 hr after a single intraperitoneal injection of 1.5 micrograms of interferon per mouse; at this time the interferons were no longer detectable in serum. Near-normal levels of cytochrome P-450 and in vitro drug metabolism activity were restored by 48 hr after a single injection of interferon. Treatment of mice with 1.5 micrograms of IFLrA/D once daily for 3 days prolonged hexobarbital sleeping time but not zoxazolamine paralysis time, whereas neither of these was influenced by treatment with IFLrA or D. The results indicate that an interferon-dependent process reduces the level of microsomal cytochrome P-450 in liver and potentiates the pharmacological actions of certain drugs in mice.