Heat-alkali treatment of synthetic alpha- and beta-melanocyte-stimulating hormones (MSH), known to cause racemization of amino acids within the peptides, results in prolongation of the darkening (melanophore dispersion) effect of these hormones on frog and lizard skins in vitro. Skins remain darkened for hours or even days if supramaximal concentrations of the racemized hormones are used. This response can be partially reversed by melatonin or noradrenaline. Heat-alkali treatment of alpha-MSH at either 60 or 97 degrees C results in a retardation of the response of the skins to the racemized peptides. In contrast, the response of frog skins to heat-alkali-treated beta-MSH is immediately enhanced and potentiated. Heat-alkali treatment also prolongs and potentiates the activity of synthetic [des-acetyl]-alpha-MSH (in contrast to the retardation effect on the natural acetylated peptide). These data suggest a role for the N-acetyl group in the retardation phenomenon. The activity of synthetic [2-D-tyrosine]-alpha-MSh is much lower than that of alpha-MSH itself, indicating that heat-alkali treatment of the hormone may produce either potentiation or partial inactivation of the peptide, depending on the site of racemization.