The role of NADPH reductase and cytochrome b5 on glutathione (GSH)-induced stimulation of P450 3A4 activity was investigated. GSH increased the Vmax of testosterone 6 beta-hydroxylation without changing the K(m) for testosterone whereas it decreased the K(m) for NADPH-P450 reductase. Addition of cytochrome b5 inhibited testosterone 6 beta-hydroxylation in the reconstituted system, depleting GSH, while it dramatically enhanced the rate of testosterone 6 beta-hydroxylation in the presence of GSH. Cumene hydroperoxide-mediated P450 3A4 activity, which is independent of NADPH-P450 reductase and cytochrome b5, was not affected by GSH. High concentration of GSH above 4 mM was inhibitory in the reconstituted systems. These results suggest that GSH increases the apparent affinity between P450 3A4 and NADPH-P450 reductase, and between P450 3A4 and cytochrome b5, but has no effect on the affinity between P450 3A4 and testosterone.