Specific binding of human beta-endorphin to NG108-15 cells is described; human beta-[Tyr27-3H2] endorphin was used as the ligand. The binding is time dependent and saturable; Kd = 0.3 nM and ka = 1.8 x 10(8) M-1 min-1. Under the conditions optimal for beta-endorphin binding, leucine-enkephalin has one-fourth to one-third as many binding sites as beta-endorphin and its affinity is 7--10% that of beta-endorphin. Monovalent and divalent cations potently inhibit binding. Trypsin, phospholipase A, and N-ethylmaleimide reduce the ability of NG108-15 cells to bind beta-endorphin. beta-Endorphin analogs are able to fully inhibit the binding of beta-[Tyr27-3H2]endorphin, although enkephalins, morphine, and naloxone inhibit only 50--80%.