Nalbuphine is a potent agonist/antagonist analgesic with a low side effect profile and low abuse potential. Previous studies have shown that nalbuphine produces predominantly agonist (analgesic) effects at kappa receptors and antagonist (morphine-reversal) effects at mu receptors in vivo. The present study was designed to localize the sites of nalbuphine binding to mu, delta and kappa opioid receptors in the central nervous system (CNS) using in vitro labeling light microscopic autoradiography. Mu, delta and kappa opioid receptors were labeled selectively using [3H]dihydromorphine, D-[3H]Ala2-D-Leu5-enkephalin and (-)-[3H]ethylketocyclazocine, respectively. In displacement studies in rat brain homogenates, nalbuphine had the highest affinity (Ki) for mu receptors (0.5 nM) with progressively lower affinities for kappa (29 nM) and delta (60 nM) opioid receptors. In autoradiographic studies in slide-mounted sections of guinea pig brain and monkey spinal cord, nalbuphine (300 nM) displaced completely the binding at mu and kappa receptors without significantly altering the binding at delta receptors. The binding of [3H]nalbuphine in slide-mounted sections of guinea pig forebrain was saturable and showed a curvilinear profile indicating the presence of two binding sites with apparent dissociation constant (Kd) values of 0.5 and 12 nM. Morphine and U-50,488H, which have high affinities for mu and kappa opioid receptors, respectively, inhibited [3H]nalbuphine binding with IC50 values of 0.9 and 10 nM, respectively. In saturation studies, morphine (50 nM) and U-50,488H (100 nM) selectively blocked the high and low affinity components of [3H]nalbuphine binding, respectively. The autoradiographic distribution of [3H]nalbuphine binding sites in the CNS corresponds well to the distribution of mu and kappa opioid receptors. In addition, CNS areas (deep layers of the cerebral cortex, laminae I and II of the spinal cord, substantia gelatinosa of the trigeminal nerve, periaqueductal gray and thalamic nuclei) that mediate analgesia contain high concentrations of [3H]nalbuphine binding sites. In summary, these data demonstrate that nalbuphine acts on mu and kappa opioid receptors and identify anatomical loci in the CNS in which nalbuphine may produce its actions.