Catalysis by all 50 Type II (microsomal) P450 enzymes, including steroidogenic P450c17, P450c21, and P450aro and hepatic drug-metabolizing enzymes requires electron donation from P450 oxidoreductase (POR). POR knockout mice are embryonic lethal, but human POR mutations cause a complex disorder of steroidogenesis. Disorders of hepatic drug metabolism in human POR deficiency have not yet been described. To understand the potential contribution of POR to pharmacogenetics, we sequenced the POR gene in 842 normal persons from 4 ethnic groups. We detected 140 single nucleotide sequence variants of which 43 were in >1% of alleles, including 15 missense mutants; this brings the total of known POR missense mutants to 35. A503V was found on 28% of alleles, varying from 19% in African Americans to 37% in Chinese Americans. We expressed all 35 missense mutants in E. coli and assayed their activities to: oxidize NADPH, reduce cytochrome c, support the 17alpha-hydroxylase and 17,20 lyase activities of bacterially expressed human P450c17, and support the metabolism of fluorogenic EOMCC by bacterially expressed human CYP1A2 and CYP2C19. These data show that there are great differences in the activities of some POR mutants depending on the electron recipient assayed; for example, Q153R causes severely impaired steroid biosynthesis in human patients and in vitro, but is a gain-of-function mutant with CYP1A2 and 2C19. A503V reduces both activities of P450c17 in half, but had no effect on CYP1A2 or 2C19. POR variants are a previously unappreciated source of genetic variation in patterns of steroid synthesis and drug metabolism.