Multiple forms of alpha-MSH were isolated from the pituitaries of the white sturgeon, Acipenser transmontanus, and the paddlefish, Polyodon spathula, following fractionation of the pituitary extracts by reversed-phase HPLC. In both species, immunoreactive forms which eluted with the same retention times as synthetic mammalian ACTH(1-13)NH2, monoacetylated alpha-MSH, and diacetylated alpha-MSH were detected. In the pituitary of the sturgeon, N-acetylated forms of alpha-MSH represented 91% of the total alpha-MSH isolated. However, in the pituitary of the paddlefish, N-acetylated forms of alpha-MSH accounted for only 45% of the total alpha-MSH isolated. The presence of N-acetylated forms of alpha-MSH in the pituitaries of representatives of the two extant genera of chondrostean fish coupled with the observation that N-acetylated forms of alpha-MSH are present in the pituitaries of cladistian and neopterygian fishes indicates that the alpha-MSH specific N-acetylation mechanism evolved prior to the radiation of the Actinopterygii. A conspicuous feature of the chondrostean alpha-MSH N-acetylation reaction was the low levels of diacetylated alpha-MSH produced relative to the levels of monoacetylated alpha-MSH. Similar observations have been made for the cladistian fish, Calamoichthys calabaricus, and the cartilaginous fish, Hydrolagus colliei. However, in holostean fishes, lungfishes, and tetrapods, the diacetylated form of alpha-MSH is the major end product produced in the intermediate pituitary. The phylogenetic implications of these observations will be discussed.