To define the hormonal criteria via genotypic proof for 3beta-hydroxysteroid dehydrogenase (3beta-HSD) deficiency in the adrenals and gonads, we investigated the type II 3beta-HSD genotype in 55 patients with clinical and/or hormonal presentation suggesting compromised adrenal with or without gonadal 3beta-HSD activity. Fourteen patients (11 males and 3 females) had ambiguous genitalia with or without salt wasting and with or without premature pubarche. One female neonate had salt wasting only. Twenty-five children (4 males and 21 females) had premature pubarche only. Fifteen adolescent and adult females had hirsutism with or without menstrual disorder. The type II 3beta-HSD gene, including the promoter region up to -1053 base, all exons I, II, III, IV, and exon and intron boundaries, was sequenced in all subjects. Eight patients had a proven or predictably deleterious mutation in both alleles of the type II 3beta-HSD gene, and 47 patients had no apparent mutation in the gene. ACTH-stimulated (1 h post iv bolus of 250 microg Cortrosyn) serum 17-hydroxypregnenolone (Delta5-17P) levels and basal and ACTH-stimulated ratios of Delta5-17P to cortisol (F) in the genotypic proven patients were unequivocally higher than those of age-matched or pubic hair stage matched genotype-normal patients or control subjects (n = 7-30 for each group). All other baseline and ACTH-stimulated hormone parameters, including dehydroepiandrosterone (DHEA) levels, ratios of Delta5-17P to 17-OHP and DHEA to androstenedione in the genotype-proven patients, overlapped with the genotype-normal patients or control subjects. The hormonal findings in the genotype-proven patients suggest that the following hormonal criteria are compatible with 3beta-HSD deficiency congenital adrenal hyperplasia (numeric and graphic reference standards from infancy to adulthood are provided): ACTH-stimulated Delta5-17P levels in 1) neonatal infants with ambiguous genitalia at or greater than 378 nmol/liter equivalent to or greater than 5.3 SD above the control mean level [95 +/- 53 (SD) nmol/liter]; 2) Tanner I children with ambiguous genitalia at or greater than 165 nmol/liter equivalent to or greater than 35 SD above the control mean level [12 +/- 4.3 (SD) nmol/liter]; 3) children with premature pubarche at or greater than 294 nmol/liter equivalent to or greater than 54 SD above Tanner II pubic hair stage matched control mean level [17 +/- 5 (SD) nmol/liter]; and 4) adults with at or greater than 289 nmol/liter equivalent to or greater than 21 SD above the normal mean level [25 +/- 12 (SD) nmol/liter]. ACTH-stimulated ratio of Delta5-17P to F in 1) neonatal infants at or greater than 434 equivalent to or greater than 6.4 SD above the control mean ratio [88 +/- 54 (SD)]; 2) Tanner I children at or greater than 216 equivalent to or greater than 23 SD above the control mean ratio [12 +/- 9 (SD)]; 3) children with premature pubarche at or greater than 363 equivalent to or greater than 38 SD above the control mean ratio [20 +/- 9 (SD)]; and 4) adults at or greater than 4010 equivalent to or greater than 221 SD above the normal mean ratio [29 +/- 18 (SD)]. Conversely, the hormonal data in the genotype-normal patients suggest the following hormonal criteria are not consistent with 3beta-HSD deficiency congenital adrenal hyperplasia: ACTH-stimulated Delta5-17P levels in children with premature pubarche up to 72 nmol/liter equivalent to up to 11 SD above the control mean level, and in hirsute females up to 150 nmol/liter equivalent to up to 12 SD above the normal female mean level [28 +/- 10 (SD) nmol/liter]; and ACTH-stimulated Delta5-17P to F ratio in children with premature pubarche up to 67 equivalent to up to 5 SD above the control mean ratio, and in hirsute females up to 151 equivalent to up to 10 SD above the normal mean ratio [32 +/- 12 (SD)]. These findings help define newly proposed hormonal criteria to accurately predict inherited 3beta-HSD deficiency.