Maximal activity of CYP3A4 is obtained using a reconstitution system consisting of NADPH-P450 reductase (CPR), dioleoylphosphatidylcholine (DOPC), an ionic detergent, and cytochrome b(5) (b(5)). The mechanism by which b(5) stimulates the catalytic activity of CYP3A4 is controversial. Recent data report that apo-cytochrome b(5) (apo-b(5)) can substitute for holo-b(5) by serving as an allosteric effector. These authors concluded that b(5) is not directly involved in electron transfer reactions to CYP3A4. We have studied the effect of apo-b(5) on the ability of purified CYP3A4 to catalyze the 6beta-hydroxylation of testosterone and horse CYP17A to catalyze the 17,20-lyase reaction. The high molecular weight form of holo-b(5) (HMW-holo-b(5)) stimulates the 6beta-hydroxylation of testosterone while the low molecular weight (truncated) form of holo-b(5) (LMW-holo-b(5)) does not. When added to the reconstituted system, HMW-apo-b(5) stimulates the activity of CYP3A4 to a level 50-60% of that obtained with HMW-holo-b(5). A similar stimulation of 17alpha-hydroxyprogesterone metabolism is seen when studying the CYP17A-catalyzed reaction. Neither LMW-holo-b(5) nor LMW-apo-b(5) stimulates the activity of CYP3A4 or CYP17A. CYP3A4 forms a complex during affinity chromatography with immobilized HMW-holo-b(5) but not with immobilized HMW-apo-b(5). Incubation of apo-b(5) with CYP3A4, using conditions required for reconstitution of enzymatic activities, results in the transfer of heme from the CYP3A4 preparation to apo-b(5), thereby forming holo-b(5). The separation of heme proteins by thiol-disulfide exchange chromatography confirms the formation of holo-b(5). A His67Ala mutant of HMW-b(5) as well as the Zn-substituted protoporphyrin derivative of HMW-b(5) do not stimulate the activity of either CYP3A4 or CYP17A. These data show that the mechanism of stimulation of CYP3A4 and CYP17A activities by apo-b(5) results from the formation of holo-b(5) by a heme transfer reaction.