Cytokine-induced release of corticotropin-releasing factor (CRF) from hypothalamic explants in vitro can be inhibited by femtomolar concentrations of α-melanocyte-stimulating hormone (α-MSH). Because the mechanism of the anticytokine action of α-MSH remains unknown, we examined if the peptide inhibits CRF release by interference with various steps in the activation of CRF release. Previous studies have shown that CRF release is induced by activation of phospholipase A2 (PLA2). Therefore, we examined the effect of α-MSH on the action of melittin (MEL), a PLA2 activator. After 60 min preincubation in Krebs-Ringer bicarbonate buffer, medial basal hypothalami were incubated for 30 min with Krebs-Ringer bicarbonate buffer or MEL with or without α-MSH (10–11 to 10–16M). CRF release into the incubation medium was measured by RIA. As reported previously none of the α-MSH concentrations used changed basal CRF release nor did any concentration of α-MSH significantly alter CRF release induced by MEL (10 μg/ml). Thus, α-MSH alters cytokine-induced CRF release at a step unrelated to the activation of PLA2. Because activation of PLA2 requires an increase in intracellular calcium ion (Ca2+) concentrations, we evaluated the effect of α-MSH on the release of CRF induced by a high concentration of potassium (56 mM). This concentration of potassium induced a 3.5-fold increase in CRF release that was not affected by α-MSH. Protein kinase C (PKC) stimulates CRF release. Consequently, we examined the effect of α-MSH on CRF release induced by phorbol myristate acetate (PMA), which in the presence of Ca2+ stimulates PKC. PMA (10–10M)stimulated CRF release. There was a bell-shaped dose-response curve with lesser stimulation at 10–11 and 10–9 M which was not significant statistically and a return to baseline levels of CRF release at concentrations of 10–8 and 10–7 M. α-MSH (10–16 to 10–13 M) completely blocked this effect, but α-MSH was ineffective at 10–12M. The results indicate that the action of α-MSH is not mediated by blocking the increase in intracellular Ca2+ or the activation of PLA2, which are required for CRF release, but instead is mediated by a blockade of the activation of PKC. We speculate that a-MSH acts on the adrenocorticotropic hormone receptor on the surface of the CRF neuron to activate it leading to blockade of the activation of PKC. We hypothesize that blockade of the action of PKC may be the basis for the anticytokine actions of α-MSH in the hypothalamus and also in the immune system.