alpha-Melanocyte-stimulating hormone (alpha-MSH1-13), a peptide derived from proopiomelanocortin, has remarkable anti-inflammatory and antipyretic activities. This peptide and a tripeptide that forms the COOH-terminal portion of the molecule (alpha-MSH11-13; Lys Pro Val) inhibit inflammation when given centrally or peripherally. Because of the similarity in their actions, the tripeptide has been presumed to be the amino acid message sequence underlying the effects of alpha-MSH1-13. To test the possibility that the two peptides occupy the same receptors, competitive binding experiments were performed with B16 mouse melanoma cells that are known to have alpha-MSH1-13 receptors. In these experiments, alpha-MSH11-13 did not inhibit binding of a radiolabelled alpha-MSH1-13 analog. This finding suggests that alpha-MSH1-13 and alpha-MSH11-13 exert their anti-inflammatory/antipyretic/anticytokine effects via stimulation of separate receptors. Because alpha-MSH inhibits the effects of several cytokines including inflammation caused by interleukin (IL)-6 and IL-8, the capacity of these cytokines to compete for alpha-MSH binding sites was tested. There was no evidence that these proinflammatory cytokines bind to alpha-MSH receptors on murine melanoma cells. Although further tests with host cells involved in inflammation are required, the latter result is the first evidence that the mechanism of anticytokine action of alpha-MSH does not depend upon peptide/cytokine competition for binding sites.