Acid extracts of the brain of a urodele amphibian, Ambystoma tigrinum, were screened with radioimmunoassays specific for enkephalin-related products and dynorphin-related products. Following Sephadex G-50 column chromatography a peak of enkephalin-sized immunoreactive material was detected near the total volume of the column. The enkephalin-sized immunoreactivity was further analyzed by reversed phase HPLC. This analysis detected peaks of authentic Met-enkephalin and Leu-enkephalin. However, the molar ratio of Met-enkephalin to Leu-enkephalin in the brain of this amphibian was approximately 80:1. These observations would suggest that the Leu-enkephalin detected in the brain of Ambystoma may be derived from a source other than the Proenkephalin precursor. Neither Met-enkephalin-RGL or Met-enkephalin-RF were detected by radioimmunoassay in brain extracts from this urodele. However, following digestion with trypsin and carboxypeptidase B, a novel peak of C-terminally extended Met-enkephalin was detected. Two peaks of Prodynorphin-related products were also detected following gel filtration chromatography. These immunoreactive forms were detected using antisera specific for α-neo-endorphin and dynorphin B(1–13). No immunoreactive forms with antigenic determinants similar to mammalian dynorphin A(1–17) or dynorphin A(1–8) were detected in this species. Reversed phase HPLC analysis indicated that the major form of urodele α-neo-endorphin eluted with the same retention time as synthetic mammalian α-neo-endorphin. Urodele dynorphin B(1–13)-related immunoreactivity eluted as a single peak, however this form did not elute with the same retention time as synthetic mammalian dynorphin B(1–13). The forms of α-neo-endorphin and dynorphin B detected in urodele and anuran amphibians appear to be very similar, however it appears that amphibian dynorphin A has diverged significantly from mammalian dynorphin A. Analysis of enkephalin-related products in the brain of Ambystoma indicates that Met-enkephalin is the major opioid produced by the urodele Proenkephalin gene, and supports the contention that urodele Proenkephalin, like anuran Proenkephalin, lack a Leu-enkephalin sequence.