Acid extracts of individual pars intermedia from the Australian lungfish, Neoceratodus forsteri, were fractionated by gel filtration chromatography and analyzed for α-melanotropin (α-MSH) immunoreactivity. In these studies a C-terminal-specific α-MSH radioimmunoassay (RIA) was used. Following gel filtration chromatography on a Sephadex G-75 column, a major peak of immunoreactive α-MSH-sized material was detected. On the average there was 338 ± 72 pmol (SD) of immunoreactive a-MSH per lungfish pars intermedia (n = 3). Following gel filtration the immunoreactive α-MSH was further analyzed by reverse-phase high-performance liquid chromatography (HPLC). Three peaks of immunoreactivity were detected. These peaks were designated Peaks l, 2, and 3. The retention times of these peaks corresponded to, respectively, mammalian ACTH(1-13)amide, N-acetyl-ACTH(1-13)-amide, and N,O-diacetyl-ACTH(1-13)amide. Peaks 2 and 3 represented approximately 95% of the immunoreactive a-MSH recovered. Analysis of immunoreactive Peaks 2 and 3 by cation-ion-exchange indicated that both peaks had a net charge of +3 at pH 2.5. Since O-acetyl groups are sensitive to high pH, Peak 3 was incubated for I hr at 37° in 0.01 N NaOH, pH 12. Under these conditions, Peak 3 eluted with the same retention time as untreated Peak 2. Collectively, these results indicate that Peaks 2 and 3 correspond to monoand diacetylated lungfish α-MSH, respectively.