We measured the concentrations of α-melanocyte-stimulating hormone (α-MSH) in various brain regions as well as in the pituitary gland and plasma of the toad Bufo speciosus during a 24-hr light:dark cycle. There was significant diurnal variation of α-MSH concentrations in the hypothalamus and brainstem. In both areas α-MSH concentrations were highest during the scotophase. Peak α-MSH concentrations in the hypothalamus were observed at 21.00 and 05.00 hr, while a single peak in α-MSH concentrations was observed in the brainstem at 21.00 hr. In contrast, peak α-MSH concentrations in the plasma were observed during the photophase at 17.00 hr, when brain concentrations of α-MSH were low. There was no significant diurnal variation observed in the pituitary content of α-MSH throughout the 24-hr light:dark cycle. These data suggest that different mechanisms control hypothalamic and pituitary α-MSH cells in the toad during the 24-hr light:dark cycle.The fact that peak α-MSH concentration were observed in the hypothalamus during the activity period of the toad is consistent with the proposed role of α-MSH peptides in learning and memory processes.