Previous studies using a variety of opiate ligands have suggested the existence of several subclasses of opiate receptors in crude membrane fractions of rat brain, and a similar diversity in bovine adrenal medulla. To examine the receptor profile of bovine adrenal medulla in detail we have studied the binding of classical ligands for mu (mu), delta (delta) and kappa (kappa) opiate receptors. [3H]naloxone ([3H]NAL), [3H]morphine ([3H]MOR), [3H]D-Ala2-D-Leu5-enkephalin ([3H]DAL) and [3H]ethyl-ketocyclazocine ([3H]EKCZ) were used as tracers; unlabeled competitors were NAL, MOR, DAL and ketocyclazocine (KCZ). In adrenal medulla [3H]NAL was specifically bound with a hierarchy of displacement NAL greater than MOR greater than KCZ much greater than DAL. No specific binding of [3H]DAL or [3H]EKCZ was found; for [3H]MOR very low levels of binding were seen, with no displacement by NAL or DAL, inconsistent displacement by KCZ and substantial displacement by MOR with an ED50 of 1.5 nM. In parallel studies rat brain membranes bound each labeled ligand with affinity and specificity consistent with previously published reports. Identical results were obtained in membranes from both tissues prepared with a preincubation step including 100 mM Na+, suggesting that the results were not influenced by occupation of binding sites by endogenous ligands. We interpret these data as supporting the existence of opiate receptors of the mu subtype in bovine adrenal medulla. We find, however, no evidence of delta or kappa sites in this tissue.