The effect of 17 beta-estradiol and testosterone on glucocorticoid secretion were studied in vitro by using dispersed inner adrenocortical cells obtained from gonadectomized female and male rats. Independently of the sex of animals, estradiol enhanced basal, but not ACTH-stimulated corticosterone (B) secretion; conversely, testosterone inhibited ACTH-stimulated, but not basal B output. HPLC analysis of steroid secreted demonstrated that estradiol induced comparable rises (53-62%) in basal pregnenolone (PREG) and total post-PREG secretion (progesterone, II-deoxycorticosterone and B). Testosterone inhibited by about 30% ACTH-stimulated PREG production and by about 54% total post-PREG secretion (B was decreased to 56% of the control value, and other steroid hormones were below the limit of sensitivity of our assay system). These findings indicate that sex hormones directly affect rat adrenocortical secretion, mainly by acting on the rate-limiting step of steroidogenesis (i.e. the conversion of cholesterol to PREG); moreover, they suggest that testosterone is also able depress the activity of the enzymes operating distally to cholesterol side-chain cleavage.