Cytochrome P450c17 is the sole enzyme catalyzing both the 17α-hydroxylation of cortisol precursors and the conversion of C21 steroids to C19 sex steroids (17,20 lyase activity). It is encoded by a single-copy gene of 8 exons spanning 7 kb on chromosome 10q24-q25. Patients with 17α-hydroxylase deficiency also have 17,20 lyase deficiency, and all of the 18 characterized mutations of the P450c17 gene affect both the 17α-hydroxylase and 17,20 lyase activities equally when these mutations are re-created in transfected cell systems. Patients with hormonal findings suggestive of isolated 17,20 lyase deficiency have been reported, but the only case studied at a molecular level had combined partial deficiencies of both activities (BBA 1139: 275, 1992). However, molecular modeling and site-directed mutagenesis studies have shown that the mutation R347A can selectively ablate the lyase activity of the rat (Mol Endo 5: 1373, 1991) or human enzyme (Mol Endo 8: 392, 1994) while retaining 80-90% of the 17α-hydroxylase activity. Recently one of us (BBM) described two pseudohermaphrodites, each with microphallus, perineal hypospadias, bifid scrotum, cryptorchidism and a blind vaginal pouch. In case 1, a 13 month old, the basal ratio of serum 17OHP to ▵4A was 0.66(nl 0.5) and following stimulation with hCG rose to 13 (nl 1.0); in case 2, a 16 year old, the basal ratio was 60 (nl 0.12) and did not rise with hCG stimulation (nl 0.30), indicating 17,20 lyase deficiency but normal 17α-hydroxylase activity in both patients (Endo Soc Abstr P3-617, 1995). Sequencing of P450c17 cDNA prepared by RT-PCR from testicular RNA revealed the mutations R347H in case 1 and R358Q in case 2. Direct sequencing of genomic DNA showed each patient is homozygous, and that the consanguineous parents of each patient are heterozygotes. These are the first cases of 17,20 lyase deficiency demonstrated at the molecular level, and marks the first time the specific location of a mutation was predicted by molecular modeling studies.