Acid extracts of the intermediate pituitaries of the gars, L. spatula and L. osseus, were fractionated by Sephadex G-50 column chromatography and analyzed by radioimmunoassay. This procedure revealed that immunoreactive forms of N-acetylated beta-endorphin- and alpha-MSH-sized material were present in equimolar amounts and represented the major end products of the POMC biosynthetic pathway in these species. Cation-exchange chromatography indicated that multiple N-acetylated forms of beta-endorphin were present in the intermediate pituitaries of the two species of gar, and that these forms differed in their net positive charge and in their apparent molecular weight. Reversed-phase HPLC analysis of the alpha-MSH-related material indicated that up to 90% of the total MSH in the pituitary of the gar was N-acetylated. Furthermore, the predominant form of alpha-MSH in both species of gar was N,O-diacetyl-ACTH(1-13)-NH2. Nearly identical results were obtained following the analysis of alpha-MSH-related peptides in the intermediate pituitary of the bowfin, A. calva. The pattern of posttranslational processing of POMC observed in the intermediate pituitaries of holostean fishes is very similar to the processing events observed in lungfishes, turtles, and mammals; hence, the processing of POMC has been remarkably conserved during vertebrate evolution.