The effect of background adaptation on the steady-state levels of alpha-melanotropin in the pars intermedia of the larval amphibian. Ambystoma tigrinum, was investigated. Acid extracts of pars intermedia obtained from light-adapted and dark-adapted animals were analyzed by radioimmunoassay following Sephadex gel filtration chromatography, reverse-phase HPLC, and Sulfopropyl Sephadex cation-ion-exchange chromatography. For both background adaptation conditions similar results were obtained. The major form of alpha-melanotropin present in the pars intermedia has the following properties: (1) an apparent molecular mass of 1.5 kDa; (2) a net charge at pH 3.5 of +4; and (3) a retention time following reverse-phase HPLC similar to that of synthetic ACTH(1-13)amide. In dark-adapted animals a minor form of alpha-melanotropin which has a net charge of +3 at pH 3.5 was also detected. The latter form represented approximately 10% of the total alpha-melanotropin immunoreactivity in the pars intermedia of dark-adapted animals. These results strongly suggest that the predominant form of alpha-melanotropin in the pars intermedia of larval A. tigrinum is a nonacetylated ACTH(1-13)amide-like polypeptide.