The concentration of MSH in serum after estrogen or progesterone injection into gonadectomized rats was measured by a biological assay. Undetectable serum MSH values were found in long-term ovariectomized (OVX) rats. After a single injection of 10 µg estradiol benzoate (EB) serum MSH exhibited a circadiam rhythm with high levels in the afternoon, while values in the morning were negative. This effect occurred for 4 days following the injection. Progesterone injection into spayed rats also resulted in an increase of serum MSH concentration, but in contrast to the changes observed after EB treatment high values were found in the morning for 3 consecutive days. Afternoon MSH levels were low but measurable. The effect of these steroids on the activity of the hypothalamic enzymes which yielded MIF or MRF upon incubation with oxytocin (OXT) was also studied. Enzymatic activity of both systems was undetectable in OVX rats and was evident after EB treatment. Progesterone increased only the activity of the system which yields MRF. In castrated male rats estrogen elevated baseline MSH levels but a circadian rhythm was not observed, whereas progesterone had no effect. These observations demonstrate a sex difference on steroid-induced MSH release.