The acute depletion of the melanocyte-stimulating hormone (MSH) concentration in the pituitary gland of rats was used as the end point for evaluating the effect of different stimuli on MSH secretion. 20 min after an i.v. injection of 2% sodium chloride (2 ml) into male rats, 60 min after vaginal stimulation of female rats at estrus, or after subjecting lactating rats to 30 min suckling, the concentration of MSH in the pituitary decreased about 50 %. The injection of the hypertonic saline also resulted in a rise in the serum melanophoretic index. The depletion of MSH was blocked in response to each of these stimuli by the injection of picrotoxin (1 mg/kg) 20 min before the stimuli were applied. However, picrotoxin failed to interfere in the MSH depletion induced either by the injection of dibenamine (30 mg/kg) or of MSH-releasing factor, suggesting that its blocking effect is exerted before either the intermediate lobe or the neurons secreting MSH-release-inhibiting factor. Treatment of the animals with p-chlorophenylalanine or the injection of methysergide (10 µg) into the 3rd ventricle prevented the drop in pituitary MSH concentration which can be induced either by hypertonic saline injection or by vaginal stimulation. This indicates that 5-hydroxytryptamine (5-HT) neurons mediate the effect of these stimuli. Further support to this view was provided by the fact that the injection of 5-HT (2 µg) into the 3rd ventricle resulted in a decrease in pituitary MSH concentration. This effect of 5-HT was also blocked by the previous injection of picrotoxin. γ-Aminobutyric acid (2 µg) injected into the 3rd ventricle produced a decrease in pituitary MSH concentration, an action which was prevented by picrotoxin but not by methysergide. These experiments suggest that the studied stimuli induce MSH release by the activation of 5-HT-containing neurons which in turn inhibit the system that tonically suppresses the secretion of MSH in the intact animals.