Since beta-endorphin is the putative endogenous ligand for epsilon-opioid receptors, the previous demonstration of saturable, high affinity beta-endorphin binding sites on bovine pineal membranes suggests the possible presence of epsilon-opioid receptors. To determine the identity of pineal beta-endorphin binding sites, the inhibition of [125I]beta-endorphin binding by ligands with varying affinities for epsilon-, mu-, delta- or kappa-opioid receptors was investigated. A high positive correlation was observed between the Ki values for these drugs to inhibit [125I]beta-endorphin binding to pineal membranes and for these drugs to bind to delta-opioid receptors but not to mu-, kappa- or epsilon-opioid receptors, demonstrating that in the pineal beta-endorphin binds to delta-opioid receptors. Both NaCl and a GTP analogue were potent inhibitors of [125I]beta-endorphin binding, providing evidence that beta-endorphin is an agonist at pineal delta-opioid receptors. These results suggest that endogenous bovine beta-endorphin may modulate pineal function.