Iodinated human β-endorphin was affinity-cross-linked to opioid receptors present in membrane preparations from bovine frontal cortex, bovine striatum, guinea pig whole brain, and rat thalamus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography revealed covalently labeled peptides of 65, 53, 41, and 38 kilodaltons (kDa). The 65-and 38-kDa peptides were present in all four tissues. The 41-kDa peptide was seen only in bovine caudate and guinea pig whole brain while the 53-kDa peptide was absent in rat thalamus. All four labeled peptides were constituents of opioid receptors since their labeling was fully suppressed by the presence of excess opiates, such as bremazocine, during binding. The distribution and levels of the labeled species in the brain tissues examined and, in earlier work, in the neuroblastoma X glioma NG 108–15 cell line suggested that the 65-kDa peptide is a binding component of μ receptors while the 53-kDa peptide is a binding subunit of δreceptors. This result was strongly supported by the finding that the labeling of the 65-kDa peptide is selectively reduced by the presence of the highly μ-selective ligand Tyr-D-Ala-Gly-(N-Me)Phe-Gly-ol (DAMGE) during binding, while the labeling of the 53-kDa peptide is selectively reduced or eliminated by the highly μ-selective ligand [D-Pen2,D-Pen5] enkephalin (DPDPE). The labeling of the 41-and 38-kDa bands was reduced by either DAMGE or DPDPE. The relationship of these lower molecular weight opioid-binding peptides to μ and δ receptors is not understood. Several possible explanations are presented.