The N-terminal acetylation of melanophore-stimulating hormone (MSH) increases the melanotropic potency of the peptide. This modification may be important in amphibians, where MSH causes skin darkening during adaptation to black background. This study examines the acetylation status of the peptide in the toad Xenopus laevis under different conditions of background adaptation. Acetylated and nonacetylated α-MSH were analyzed by high-performance liquid chromatography and quantified by radioimmunoassay. The acetylation status of α-MSH was analyzed in tissue, in plasma and in media obtained from in vitro incubation of neurointermediate lobe tissue. Nonacetylated MSH is the major form of α-MSH in tissue from both black- and white-background-adapted animals. In plasma of black-adapted animals only acetylated α-MSH could be detected. Plasma MSH levels of white-adapted animals were barely detectable. Analysis of peptides secreted during in vitro incubations of neurointermediate lobe tissue from black-adapted animals showed that the relative contribution of α-MSH to the immunoreactive profile was considerably enhanced, which supports the concept that acetylation of MSH in Xenopus is associated with the secretory process. Acetylation capacity of tissue from white-adapted animals was much lower and only after several days on black background was full capacity acquired. It is suggested that de novo biosynthesis of acetylation enzymes may be necessary for the acquisition of the acetylation capacity. Transfer of black animals to white background caused a rapid decrease in acetylation capacity, which suggests that factors involved in the rapid inhibition of secretion might also regulate acetylation. This finds support in the observation that dopamine was effective in partially inhibiting the acetylation capacity of this tissue. It is concluded that the acetylation of MSH in the pars intermedia of X. laevis is a regulated process related to physiological parameters concerning background c