Using prototypic ligands for each type of opioid receptors (mu, delta, kappa, and sigma) as well as compounds derived from each class of endogenous opioid peptides (beta-endorphin, enkephalins, and dynorphins), we have undertaken the characterization of adrenomedullary opioid binding sites. The specific binding of [3H]etorphine ([3H]ET) to a membrane preparation of bovine adrenal medulla was greatly increased when the incubation temperature was raised from 22 to 37 degrees C. Characterization of the opioid binding sites was obtained at 37 degrees C with [3H]ET (nonspecific opioid ligand), [3H]ethylketocyclazocine ([3H]EKC; kappa), [3H]dihydromorphine ([3H]DHM; mu), [3H]-[D-Ala2,D-Leu5]enkephalin ([3H]DADLE: delta), and N-[3H]allylnormetazocine ([3H]SKF-10047; sigma) in the absence or presence of blocking agents for cross-reacting receptors. [3H]ET had a high affinity binding site (KD = 0.98 nM) with a Bmax of 119 pmol/g protein. All the other opioid compounds showed biphasic saturation curves with KD ranging from 0.6 to 1.29 nM for the high affinity binding site and from 2.49 to 12.1 nM for the low affinity binding site. The opioid mu-receptor was characterized by the high affinity binding site for [3H]DHM (KD = 1.29 nM; Bmax = 38 pmol/g protein). Blockade of the cross-reacting receptor sites for [3H]EKC, [3H]DADLE, and [3H]SKF-10047 revealed the presence of kappa (KD = 0.66 nM; Bmax = 12 pmol/g protein), kappa 2 (benzomorphan site; KD = 11.1 nM; Bmax = 56 pmol/g protein), delta (KD = 0.67 nM; Bmax = 4.7 pmol/g protein), and sigma (KD = 4.54 nM; Bmax = 32 pmol/g protein) opioid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)