The recombinant fusion protein containing the heme domain of human P450 3A4 and the flavin domains of rat NADPH-cytochrome P450 (P450) reductase (rF450[mHum3A4/mRatOR]L1) requires both phospholipid and detergent as well as cytochrome b5 (b5) for the NADPH-dependent catalysis of the 6 beta-hydroxylation of testosterone. NADPH oxidation results in the formation of hydrogen peroxide in the presence or absence of phospholipid and detergent. NADPH oxidation and hydrogen peroxide formation are inhibited by the addition of b5 and stimulated greater than 3-fold by the addition of testosterone. Marked differences in the ability of various phospholipids to support the P450-dependent 6 beta-hydroxylation of testosterone by the fusion protein were seen. Addition of a 4-fold excess of purified NADPH-P450 reductase, in the presence of phospholipid, detergent, and b5, stimulates the rate of testosterone 6 beta-hydroxylation approximately 10-fold, providing turnover rates as high as 80 min-1 for P450 3A4. Approximately 30% of the rate of hydrogen peroxide formation is not sensitive to inhibition by the P450 inhibitor ketoconazole, suggesting hydrogen peroxide (or superoxide anion) formation directly from the reduced flavin domains of the fusion protein. It is proposed that the stimulation of NADPH oxidation observed following the addition of testosterone to the fusion protein may serve as a useful means of monitoring the interaction of other substrates with this P450 and thereby permit the rapid screening of chemicals to evaluate their potential metabolism by a human P450.