The hepatic microsomal metabolism of methoxyflurane in rabbits is markedly stimulated by treatment with phenobarbital. However, the increased rate of metabolism cannot be completely accounted for by the activity of the purified phenobarbital-inducible cytochrome P-450 isozyme 2, even in the presence of cytochrome b5. The discovery of a second hepatic phenobarbital-inducible cytochrome P-450, isozyme 5, led us to undertake experiments to determine in hepatic and pulmonary preparations the portion of microsomal metabolism of methoxyflurane catalyzed by cytochrome P-450 isozymes 2 and 5. We report herein that isozyme 2 accounts for 25% and 29%, respectively, of the O-demethylation of methoxyflurane in hepatic microsomes from untreated and phenobarbital-treated rabbits, and for 25% of the methoxyflurane metabolism in pulmonary microsomes. Results for isozyme 5 indicate that it catalyzes 19% and 27% of methoxyflurane metabolism in control and phenobarbital-induced liver, and 47% of O-demethylation in the lung. In summary, we demonstrate that methoxyflurane O-demethylation in lung, phenobarbital-induced liver, and control liver microsomes is catalyzed by cytochrome P-450 isozymes 2 and 5. Results with purified cytochrome P-450 isozyme 5 are consistent with those obtained using microsomal preparations. Furthermore, metabolism of methoxyflurane by purified isozyme 5 is markedly stimulated by cytochrome b5. A role for cytochrome b5 in cytochrome P-450 isozyme 5-catalyzed metabolism of methoxyflurane was also demonstrated in microsomes. Antibody to isozyme 5 was unable to inhibit methoxyflurane metabolism in the presence of maximally inhibiting concentrations of cytochrome b5 antibody.(ABSTRACT TRUNCATED AT 250 WORDS)