The present study investigated the interactions between cytochrome P450 (P450) enzymes and the NADPH:cytochrome oxidoreductase (OR) in the microsomal membrane. Microsomes containing human cytochrome P450 2A6 (h2A6) coexpressed with human OR (hOR) via a baculovirus expression system displayed coumarin hydroxylase activity with apparent Km and Vmax values of 0.41 microM and 4.05 nmol/min/nmol P450, respectively. Incorporation of purified rat liver cytochrome b5 (b5) into the microsomes increased the Vmax 2.5-fold, but did not affect the Km. The N-nitrosodimethylamine (NDMA) demethylase activity of human cytochrome P450 2E1 (h2E1) coexpressed similarly was characterized previously. Coumarin was shown not to be a substrate nor an inhibitor of h2E1, and NDMA was not a substrate nor an inhibitor of h2A6. In microsomes containing h2A6, h2E1, and hOR (M-h2A6-h2E1-hOR) obtained from a triple expression system, the two P450 enzymes were shown to compete with each other for interaction with hOR. In incubations with M-h2A6-h2E1-hOR, the presence of a h2A6 substrate (coumarin) decreased NDMA demethylase activity by a maximum of 47%, and the presence of a h2E1 substrate (NDMA) decreased coumarin hydroxylase activity by a maximum of 19%. This substrate-induced competition between h2A6 and h2E1 was decreased by the addition of purified b5. In the absence of a substrate, the NADPH-dependent H2O2 formation was high in both M-h2A6-h2E1-hOR and M-h2E1-hOR, but low in M-h2A6-hOR. The addition of NDMA had little effect on the H2O2 formation in M-h2A6-h2E1-hOR and M-h2E1-hOR. The addition of coumarin, however, slightly decreased H2O2 formation in M-h2A6-h2E1-hOR, but drastically increased H2O2 formation in M-h2A6-hOR. These results suggest that the presence of a h2A6 substrate decreased the electron flow to h2E1 in M-h2A6-h2E1-hOR. The activities of coumarin hydroxylase and NDMA demethylase of M-h2A6-h2E1-hOR were decreased and increased, respectively, by an increase in ionic strength. The ionic strength, however, did not drastically change the substrate-induced competition between h2A6 and h2E1 for hOR. The results demonstrate the usefulness of the coexpression system for mechanistic studies and illustrate that the interaction of monooxygenase enzymes in the microsomal membrane is regulated by the presence of substrates and b5.