The in vitro biosynthesis of estrogens and androgens by gonadal tissues of the ovotestes was studied in three siblings with familial true hermaphrodism and correlated with daily steroid and gonadotropin plasma levels. The probands were 15, 13, and 11 years old with normal male phenotype and external genitalia, grade III hypospadias, bilateral scrotal ovotestes, gynecomastia, and no uterus or fallopian tubes. Their karyotypes were 46XX both in peripheral lymphocytes and in gonadal fibroblasts, and no Y chromosome fluorescence was observed. A fusiform biopsy of each gonad was obtained, and the testicular and ovarian structures were excised and incubated for five days at 37 C with 3.8 muCi of 7alpha3H dehydroepiandrosterone, in Eagle's growth media, 95% O2 and 5% CO2. After standard procedures, four extractions with methylene chloride were performed, and the residue was assayed using Sephadex LH no. 20 chromatography. Testosterone (T), delta4androstenedione (delta 4), 5alphadehydrotestosterone (5alphaDHT), estrone (E1), estradiol-17 beta (E2) and estriol (E3) were measured. Also, during 16 consecutive days daily venous samples were obtained, and FSH, LH, E2, progesterone (P), and testosterone (T) were determined. The predominant steroids formed in vitro were estrogens, mainly E1 by either the testicular or ovarian structures. In the 11-year-old subject, the ovotestes were less active than in his oldest siblings. The patterns of androgen production showed that T was the principal androgen formed, followed by delta4 and minimal amounts of 5alphaDHT. The daily plasma hormonal profile resembled more closely a female pattern, specially in the 15 and 13-year-old patients. It is suggested that the ovotestes of these siblings had the enzymatic mechanisms necessary for estrogen and androgen biosynthesis, mainly E1 and T using a preferential metabolic pathway via androstenedione. Furthermore, it seems that the testicular structures had a greater capability to synthesize estrogens than the ovary.