Using adjacent section autoradiography-immunocytochemistry, the distribution of [3H]naloxone binding sites was studied in relation to neuronal systems containing [Leu]enkephalin, dynorphin A, or beta-endorphin immunoreactivity in rat brain. Brain sections from formaldehyde-perfused rats show robust specific binding of [3H]naloxone, the pharmacological (mu-like) properties of which appear unaltered. In contrast, specific binding of the delta ligand [3H]D-Ala2,D-Leu5-enkephalin was virtually totally eliminated as a result of formaldehyde perfusion. Using adjacent section analysis, we have noted associations between [3H]naloxone binding sites and one, two, or all three opioid systems in different brain regions; however, in some areas, no apparent relationship could be observed. Within regions, the relationship was complex; for example, in caudate-putamen, patches of opioid receptors did not correspond to the distribution of enkephalin immunoreactivity, but there was a correspondence between subcallosal streaks of binding sites and enkephalin. The complexity of the association between [3H]naloxone binding sites and the multiple opioid systems, and previous reports of colocalization of mu and kappa receptors in rat brain, are inconsistent with a simple-one-to-one relationship between a given opioid precursor and opioid receptor subtype. Instead, since differential processing of the three precursors gives rise to peptides of varying receptor subtype potencies and selectivities, the multiple peptide-receptor relationships may point to a key role of post-translational processing in determining the physiological consequences of opioid neurotransmission.