Rifampin is a semisynthetic antibiotic which is known to alter hepatic cytochrome P-450 mediated drug metabolizing enzymes. Using benzo(a)pyrene as the substrate we have shown that rifampin acts as a non-competitive inhibitor of hepatic microsomal aryl hydrocarbon hydroxylase in vitro at or below 0.10 mM and that it is also a non-competitive inhibitor of intestinal aryl hydrocarbon hydroxylase at or below 0.075 mM. These results suggest that the administration of rifampin with other drugs may result in altered drug biotransformation.