Acid extracts of the brain of the holostean fish Lepisosteus platyrhincus and the forebrain of the dipnoan fish Neoceratodus forsteri were separately fractionated by Sephadex G-50 column chromatography. For both species, Met-enkephalin-related immunoreactivity was detected coeluting with the total volume internal standard. Higher-molecular-weight Met-enkephalin-containing immunoreactive peaks were not detected in these chromatographs. Furthermore, immunoreactive forms with antigenic determinants identical to mammalian dynorphin A(1-17), dynorphin A(1-8), α-neo-endorphin, or dynorphin B(1-13) were not detected in either species. Reverse-phase HPLC analysis of enkephalin-sized immunoreactive material indicated the presence of authentic Met-enkephalin and Leu-enkephalin in the extracts of both species. In the brain of L. platyrhincus the molar ratio of Met-enkephalin to Leu-enkephalin was approximately 3:1, whereas, the molar ratio of these enkephalins in the forebrain of N. forsteri was approximately 14:1. C-terminally extended forms of Met-enkephalin were also detected in the extracts of both species. These results suggest that the ancestral proenkephalin gene of both actinopterygian and sarcopterygian fish contained both the Met-enkephalin and Leu-enkephalin sequences.