We have incorporated cytochrome P450 3A4 (CYP3A4) and NADPH-cytochrome P450 reductase (CPR) into liposomes with a high lipid:protein ratio by an improved method. In the purified proteoliposomes CYP3A4 binds testosterone with Kd,app = 36 (±6) μM and Hill coefficient = 1.5 (±0.3), and 75 (±4) % of the CYP3A4 can be reduced by NADPH in the presence of testosterone. Transfer of the first electron from CPR to CYP3A4 was measured by stopped-flow, trapping the reduced CYP3A4 as its FeII-CO complex and measuring the characteristic absorbance change. Rapid electron transfer is observed in the presence of testosterone, with the fast phase, representing 90% of the total absorbance change, having a rate of 14 (±2) s-1. Measurements of the first electron transfer were performed at various molar ratios of CPR:CYP3A4 in proteoliposomes; the rate was unaffected, consistent with a model in which first electron transfer takes place within a relatively stable CPR-CYP3A4 complex. Steady-state rates of NADPH oxidation and of 6β-hydroxytestosterone formation were also measured as a function of the molar ratio of CPR:CYP3A4 in the proteoliposomes. These rates increased with increasing CPR:CYP3A4 ratio, showing a hyperbolic dependency indicating an apparent dissociation constant of ~0.4 mM. This suggests that the CPR:CYP3A4 complex can dissociate and reform between the first and second electron transfers.