In vitro receptor autoradiography was used to determine the localization of binding sites with high selectivity for 4,5-epoxymorphinans (lambda sites) in rat brain slices. [3H]Naloxone was used both to label a combination of mu and lambda sites (ligand alone) and to selectively label lambda sites (ligand plus 300 nM diprenorphine added to saturate mu, delta and kappa sites), using incubation conditions optimized for binding to lambda sites. Computerized densitometric analysis confirmed the ligand specificity profile and ionic sensitivity seen for lambda sites in previous homogenate studied. The proportion of mu and lambda sites labeled by [3H]naloxone varied among different brain regions examined. The labeling in the cerebellum, the accessory olfactory bulb and the mossy fiber path of the hippocampus was almost entirely lambda in nature under the conditions employed. A number of regions showed varying proportions of lambda and mu sites, including the cortex, the amygdala, substantia gelatinosa and several thalamic nuclei. Regions labeled by [3H]naloxone containing little or no lambda binding included the striatal patches, the habenula, the substantia nigra and the inferior colliculus. Identification of brain regions with unique lambda site content may facilitate the search for its potential biological function.