The distribution of delta-opioid binding sites was studied by quantitative autoradiography in rat brain and spinal cord using the highly selective ligand [125I][D.Ala2]deltorphin-I. The binding properties of [125I][D.Ala2]deltorphin-I were investigated by microdensitometry of autoradiographic films with the aid of a computer-assisted image-analysis system. [125I][D.Ala2]deltorphin-I appeared to interact with a single class of sites in all brain areas (KD = 0.9 nM). In 23 regions tested, whatever the delta site concentration, DTLET, a delta agonist, appears to be 2 orders of magnitude more effective than DAGO, a mu agonist, in inhibiting specific [125I][D.Ala2]deltorphin-I binding. The distribution of [125I][D.Ala2]deltorphin-I sites is globally consistent with that of other delta ligands and does not support the existence of a delta-receptor subtype recognized by [D.Ala2]deltorphin-I. [125I][D.Ala2]deltorphin-I binding sites were highly confined, exhibiting selective localization in the neocortex and a diffuse pattern in the striatum, accumbens nucleus, claustrum, layer of bulb, amygdaloid nucleus, pontine nuclei, and inferior colliculus. In several areas a rostro-caudal gradient of site concentration was indicated. [D.Ala2]deltorphin-I binding sites were also present in the substantia gelatinosa at all levels of the spinal cord and, unexpectedly, in deeper laminae and the ventral horn. These results demonstrate the ability of [125I][D.Ala2]deltorphin-I to characterize low concentrations of binding sites and to reveal new localizations of delta receptors.