The metabolism of nitrosamines by microsomal cytochrome P-450 (P-450) isozymes was studied in a reconstituted monooxygenase system. P-450 LM2, LM3a, LM3b and LM3c, LM4, and LM6 were purified, respectively, from the livers of phenobarbital-treated, ethanol-treated, untreated, isosafrole-treated, and imidazole-treated rabbits. Of these isozymes, LM3a had the highest N-nitrosodimethylamine demethylase (NDMAd) activity with a Km of 2.9 mM and Vmax of 9.3 nmol/min/nmol. LM2, LM4, and LM6 exhibited NDMAd activity only at high N-nitrosodimethylamine concentrations, and isozymes LM3b and LM3c had poor activity even at the highest substrate concentrations examined. LM2, however, was more active than LM3a in the metabolism of N-nitrosomethylaniline. With each isozyme (LM3a or LM4), only one Km for NDMAd was observed, whereas with rabbit liver microsomes, multiple Km of 0.07, 0.27, and 36.8 mM were obtained. P-450 isozymes also catalyzed the denitrosation of nitrosamines at rates comparable to or lower than the demethylation, and the ratio of these two reactions was different with different nitrosamines. 2-Phenylethylamine and 3-amino-1,2,4-triazole, which were believed previously to affect NDMAd by mechanisms independent of P-450, were shown to be potent inhibitors of P-450-dependent NDMAd. These results further establish the role of P-450 isozymes in the metabolism of nitrosamines and indicate that LM3a is apparently responsible for the increased N-nitrosodimethylamine metabolism associated with ethanol treatment.