The capacity of cytochrome b(5) (b(5)) to influence cytochrome P450 activities has been extensively studied and physiologically validated. Apo-b(5) enhances the activities of CYP3A4, CYP2A6, CYP2C19, and CYP17A1 but not that of CYP2E1 or CYP2D6, suggesting that the b(5) interaction varies among P450s. We previously showed that b(5) residues E48 and E49 are required to stimulate the 17,20-lyase activity of CYP17A1, but these same residues might not mediate b(5) activation of other P450 reactions, such as CYP2E1-catalyzed oxygenations, which are insensitive to apo-b(5). Using purified P450, b(5), and reductase (POR) in reconstituted assays, the D58G/D65G double mutation, of residues located in a hydrophilic α-helix of b(5), totally abolished the ability to stimulate CYP2E1-catalyzed chlorzoxazone 6-hydroxylation. In sharp contrast, the D58G/D65G double mutation retained the full ability to stimulate the 17,20-lyase activity of CYP17A1. The D58G/D65G double mutation competes poorly with wild-type b(5) for binding to the CYP2E1·POR complex yet accepts electrons from POR at a similar rate. Furthermore, the phospholipid composition markedly influences P450 turnover and b(5) stimulation and specificity, particularly for CYP17A1, in the following order: phosphatidylserine > phosphatidylethanolamine > phosphatidylcholine. The D58G/D65G double mutation also failed to stimulate CYP2C19-catalyzed (S)-mephenytoin 4-hydroxylation, whereas the E48G/E49G double mutation stimulated these activities of CYP2C19 and CYP2E1 equivalent to wild-type b(5). We conclude that b(5) residues D58 and D65 are essential for the stimulation of CYP2E1 and CYP2C19 activities and that the phospholipid composition significantly influences the b(5)-P450 interaction. At least two surfaces of b(5) differentially influence P450 activities, and the critical residues for individual P450 reactions cannot be predicted from sensitivity to apo-b(5) alone.