Human P450c17 catalyzes the 17α-hydroxylation of pregnenolone to 17OH pregnenolone and of progesterone to 17α-OH progesterone; the same P450c17 enzyme also catalyzes 17,20 lyase activity on the same active site, converting 17OH-pregnenolone to DHEA. Rodent and porcine P450c17 also catalyze 17,20 lyase activity with Δ⁴ substrates, converting 17OH-progesterone to Δ⁴ androstenedione, but human P450c17 catalyzes this reaction very inefficiently, so that virtually all human C19 sex steroids are made via 17OH pregnenolone and DHEA. P450c17 is encoded by a single gene and a single species of mRNA. Many mutations of this gene have been described, but until recently all of these either entirely eliminated both 17Δ-hydroxylase and 17,20 lyase activity, or affected each activity equivalently. We have identified and characterized the first patients with P450c17 mutations that selectively ablate 17,20 lyase activity while retaining 17Δ-hydroxylase activity. Through a combination of enyzmologic experiments in transfected mammalian cells and in genetically manipulated yeast, plus a computer model of human P450c17, we have proven that the responsible mutations, R347H and R358Q lie in the redox-partner binding site of P450c17. This site, through which P450c17 interacts with P450 oxidoreductase to receive the electrons needed for catalysis, can be allostericly influenced by cytochrome b_5. These two mutations have contributed substantially to our understanding of the mechanisms by which 17α-hydroxylase and 17,20 lyase activities are regulated independently, and thus have contributed to the study of regulated 17,20 lyase activity in adrenarche, aging, and the polycystic ovary syndrome.