Opioid peptides have been shown to modulate various parameters of both the humoral and cellular arms of the immune system. The modulatory capacity of the peptides can often be substantially reduced in the presence of naloxone, an opioid receptor antagonist, indicating a classical ligand-receptor interaction. In order to characterize these interactions further, we investigated the characteristics of opioid receptors on a macrophage cell line, P388d1. A δ-class opioid receptor was found with an M(r) of 58,000. We also identified opioid receptors on MOLT-4 (T-cell) and IM-9 (B cell) cell lines as well as thymocytes and T cell- and B cell-enriched populations. Using the central (brain) κ-selective agonist, U-69,593, it was also determined that P388d1 cells possess κ-like opioid receptors. Scatchard analysis of the binding of [3H]U-69,593 revealed a single population of sites with a dissociation constant of 17 ± 3 (S.E.M.) nmol/l and a total number of binding sites of 53.8 ± 1.0 (S.E.M.) fmol/106 cells. Moreover, the racemic κ-selective agonist U-50,488H was able to displace 50% of [3H]U-69,593 binding at 8.0 nmol/l, whereas other opioid ligands such as [Met]-enkephalinamide (δ-selective) and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (μ-selective) were ineffective displacers of [3H]U-69,593 except at high concentrations.