OBJECTIVE: To determine the clinical and molecular genetic characterization of two Japanese patients with 17alpha-hydroxylase deficiency, we analysed the 17alpha-hydroxylase/17,20-lyase gene (CYP17). Next, to clarify the mechanism of hypoaldosteronism in 17alpha-hydroxylase deficiency, we analysed the expression of aldosterone synthase (CYP11B2) messenger RNA and sequenced CYP11B2 in these patients. PATIENTS: Patient 1 (46 XY), phenotypically female, sought medical attention for hypertension, amenorrhea and infantile genitalia. Patient 2 (46 XX), phenotypically female, presented for hypertension and amenorrhea. Hormonal data in both patients showed decreased levels of sex steroids, cortisol, aldosterone and plasma renin activity and extreme elevation of deoxycortisol. DESIGN: Direct sequencing of CYP17 and CYP11B2 was performed using genomic DNA from the patients. An expression studies of mutated forms of CYP17 was performed using COS-1 cells. The expression of CYP11B2 messenger RNA in mononuclear leucocytes (MNLs) of these patients and normal subjects was measured using the competitive polymerase chain reaction METHOD: The effect of renin secretion stimulation on the levels of CYP11B2 messenger RNA in MNLs of normal subjects was also studied. RESULTS: We detected two novel genetic defects in 17alpha-hydroxylase. Sequence analysis revealed one base pair deletion (T) at codon 243 in exon 4 in patient 1. CYP17 in patient 2 contained a point mutation (C to T) at position 415 in exon 8. Transfected cells of mutant from patient 1 had no 17alpha-hydroxylase or 17,20-lyase activity. The R415C mutant protein showed very weak activity of 17alpha-hydroxylase or 17,20-lyase activity. In the renin secretion stimulating test, the increase in CYP11B2 messenger RNA levels in MNLs was parallel with that of plasma aldosterone concentration. The expression of CYP11B2 mRNA in NMLs of these patients was lower compared to controls. No mutations in CYP11B2, including the 5' flanking region, were found. CONCLUSIONS: These results indicate that the novel mutations of the CYP17 gene found in these patients inactivate cytochrome P450c17 function, and that hypoaldosteronism in these patients may be partly explained by a decreased activity of aldosterone synthase, which is regulated at the transcriptional level.