In studies of a 6-yr-old boy and his non-HLA identical 8-yr-old sister, we demonstrated 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) deficiency in the biosynthetic pathways of glucocorticoids and androgens, but not mineralocorticoids. The sister did not manifest abnormal genital development at birth, but developed premature adrenarche at the age of 4 yr, with clitoromegaly and advanced bone age. The brother had perineal hypospadias at birth and developed premature adrenarche at the age of 6 yr. In both siblings, baseline and ACTH-stimulated delta 5 steroids were markedly elevated. The baseline and ACTH-stimulated ratios of delta 5 to delta 4 steroids remained extremely high, and all steroids promptly suppressed with dexamethasone (DEX). Normal baseline PRA and serum and urinary aldosterone (Aldo) levels increased after stimulation with a low Na+ diet. Renal Na+ conservation was normal after dietary Na+ deprivation with and without DEX administration. The PRA to pH 1 Aldo ratio remained normal with normal and low Na+ diets, regardless of DEX administration, indicating normal glomerulosa function with renin stimulation. In both siblings, ACTH increased PRA and Aldo levels, maintaining the PRA to pH 1 Aldo ratio unchanged from the baseline value. In contrast, in control children, PRA was suppressed, while Aldo increased, resulting in a fall of the PRA to pH 1 Aldo ratio. The increase in PRA with exogenous ACTH in these siblings suggests there may be an ACTH-stimulable mineralocorticoid antagonist. During prolonged DEX administration, hCG administration caused a slight increase in 17-hydroxypregnenolone and dehydroepiandrosterone in both the siblings, while testosterone (T) rose poorly in the brother, and estradiol did not rise at all in the sister. These results suggest the possibility of a deficiency of 3 beta-HSD in the gonads as well as the adrenals. After [3H]dehydroepiandrosterone iv infusion, there was normal conversion to [3H]-conjugated testosterone glucuronide, suggesting the presence of normal peripheral 3 beta-HSD activity. We propose that in these siblings, there is a deficiency of 3 beta-HSD in the adrenal zona fasciculata and zona reticularis, whereas 3 beta-HSD activity is intact in the zona glomerulosa. In addition, in these siblings, 3 beta-HSD deficiency was present in the gonads, while peripheral 3 beta-HSD activity appeared to be intact. These cases demonstrate further the heterogeneity of congenital adrenal hyperplasia due to 3 beta-HSD deficiency.