Using specific radioimmunoassays(RIAs) for methionine-enkephalin(Met-Enk), leucine-enkephalin(Leu-Enk), methionine-enkephalin-Arg6-Gly7-Leu8 (Met-Enk-Arg-Gly-Leu) and methionine-enkephalin-Arg6-Phe7 (Met-Enk-Arg-Phe), we studied the regional distribution of these opioid peptides in human and bovine brains. Met-Enk-Arg-Gly-Leu was distributed in parallel with Met-Enk, Leu-Enk and Met-Enk-Arg-Phe in human and bovine brains. The ratios of molar concentrations of these peptides are almost constant in various regions of human and bovine brains and similar to the ratio of these peptides contained in preproenkephalin A. Gel exclusion chromatography and HPLC coupled with respective RIAs showed the existence of authentic peptides without any detectable high molecular weight forms. These results indicate the parallel distribution of Met-Enk-Arg-Gly-Leu with Met-Enk, Leu-Enk and Met-Enk-Arg-Phe in various regions of human and bovine brains and further suggest that these opioid peptides are derived from the same precursor as that in the adrenal medulla and that the processing of preproenkephalin A is almost complete in human and bovine brains.