NADPH-cytochrome P450 reductase (CPR) and cytochrome-b(5) (b(5)) together with NADH-b(5) reductase (b(5)R) play important roles in cytochrome P450 3A-mediated drug metabolism via electron transfer. However, it is not clear whether variability in expression of these accessory proteins contributes to the known interindividual variability in CYP3A activity. CPR and b(5) were measured in human liver microsomes (HLMs) by spectrophotometry and immunoblotting. HLMs from elderly (>or=46 years) male donors (n=11) averaged 27% (P=0.034) and 41% (P=0.011) lower CPR levels than young (<or=45 years) male donors (n=21) for spectrophotometric and immunoblot values, respectively. Similarly, HLMs from elderly male donors averaged 43% (P=0.034) and 47% (P=0.011) lower b(5) levels than young male donors for spectrophotometric and immunoblot values, respectively. alpha-Lipoic acid and 6-propyl-2-thiouracil were evaluated for selectivity of inhibition of CPR and b(5)R activities, respectively, using recombinant enzymes and HLMs, as well as for effects on CYP3A-mediated triazolam hydroxylation in HLMs with either NADH or beta-NADPH. The results indicate that both compounds are relatively nonselective inhibitors of CPR and b(5)R activities. Finally, we used multivariate regression analysis and showed that variability in CPR or b(5) expression between HLMs does not contribute significantly to variability in CYP3A-mediated midazolam hydroxylation. Consequently, while aging is associated with decreased CPR and b(5) expression in human livers, this effect does not contribute to CYP3A variability.