Release of α-MSH from rat hypothalamic slices was characterized with respect to ionic requirements and possible diurnal variations using a sensitive radioimmunoassay. Addition of 47 mM KCl to the superfusion medium resulted in a twofold increase in α-MSH release compared to spontaneous release. Removal of calcium from the superfusion medium abolished the potassium-evoked release of α-MSH. This supports the concept that α-MSH functions as a neurotransmitter or neuromodulator in the hypothalamus. Both spontaneous and potassium-induced α-MSH release were related to diurnal variation. Marked release from the slices was observed at 10.10 h, corresponding to a peak in the α-MSH concentration in the hypothalamus  and to a low level of α-MSH in the blood. Contrarily, no significant release from the hypothalamus was obtained at 17.00 h when hypothalamic α-MSH content was low, but blood levels exhibited a peak. These findings suggest that there are differences in the regulation of the α-MSH from the pituitary and that in the hypothalamus.