A radioimmunoassay for 17-OH-pregnenolone was developed and applied to the study of this steroid in human plasma. Steroids that cross-react with the antiserum could be readily separated from 17-OH-pregnenolone; therefore the assay was highly specific. In normal subjects plasma 17-OH-pregnenolone is mainly of adrenal origin. It was largely suppressible with dexamethasone, readily responsive to ACTH and followed a diurnal rhythm similar to that of plasma fluorogenic corticosteroids with which there was a significant correlation (p < 0.001). Testicular secretion of 17-OH-pregnenolone could be inferred from the observation that while dexamethasone suppressed plasma concentrations incompletely combined treatments with dexamethasone and methyltestosterone caused the steroid to disappear completely. In contrast, women receiving dexamethasone alone had similar levels of 17-OH-pregnenolone to women receiving dexamethasone plus estrogen. Ovarian secretion of 17-OH-pregnenolone was suggested by the finding of significantly (p < 0.05) lower levels of plasma 17-OH-pregnenolone in women receiving estrogen than in untreated woman. This difference was not seen in postmenopausal women. Treatment of premenopausal women with human chorionic gonadotropin resulted in small increases in plasma 17-OH pregnenolone. Furthermore plasma 17-OH-pregnenolone was significantly (p < 0.001) higher during the follicular than during the luteal phase of the menstrual cycle. However, the ovarian contribution of 17-OH-pregnenolone to plasma (0.2 ng/ml or less) was probably inadequate to account for the phasic variation observed. The mechanism underlying the changes in plasma levels of 17-OH-pregnenolone seen during the menstrual cycle is unclear.