Sex differences in the distribution of free and esterified cholesterol in the mitochondria and lipid droplets of the rat adrenal glands, as well as their dependence on gonadal hormones were studied. For these purposes intact, gonadectomized and gonadectomized-testosterone or estradiol replaced rats were employed. The concentration of free cholesterol [FC] as well as esterified cholesterol [EC] in full homogenates of decapsulated glands was higher in male than in female rats. Neither orchiectomy nor testosterone replacement had an effect on FC and EC concentration. Ovariectomy increased FC and EC concentration in full adrenal homogenates, an effect reversed by estradiol replacement. Similar changes were found in the lipid droplets. The concentration of FC and EC in the adrenal mitochondria was higher in male than in female rats. Orchiectomy increased the concentration of FC and decreased the concentration of EC. Testosterone administration resulted in further increase in FC concentration and restored EC concentration to the level of the control group. On the other hand ovariectomy increased FC and EC concentration, an effect reversed by estradiol replacement. Results obtained clearly showed that sex differences in FC and EC concentration in rat adrenal gland depend mainly on estradiol which lowers FC and EC concentration in the adrenal lipid droplets and mitochondria.