By means of autoradiography we have studied the cellular localization of binding sites for [3H]neurotensin and its nonpeptide receptor antagonist [3H]SR-48692 in explant cultures of rat neocortex, striatum, brain stem and spinal cord. Binding sites for the peptide and its antagonist were observed on a great number of astrocytes in all CNS regions studied. Simultaneous staining of the cultures with a monoclonal antibody against glial fibrillary acidic protein has shown that the labelled cells in the outgrowth zone of the cultures were glial fibrillary acidic protein-positive and could therefore be identified as astrocytes. In addition to astrocytes, many neurons and outgrowing nerve fibres were labelled by the radioligands. Binding of [3H]neurotensin and [3H]SR-48692 (10(-8)M) to neurons and glial cells was markedly reduced or inhibited by the unlabelled compounds at high concentration (10(-6)M), suggesting "specific" binding of the radioligands. Electrophysiological studies have shown that addition of neurotensin to the bathing solution caused a hyperpolarization of the majority of astrocytes tested. There was a dose-response relationship between the magnitude of the hyperpolarization and the concentration of the peptide (10(-10)-10(-7)M); 10(-10)M being the threshold concentration. The specificity of the action of neurotensin was confirmed by the selective nonpeptide neurotensin receptor antagonist SR-48692 which reversibly blocked or markedly reduced the hyperpolarization by the peptide on all astrocytes tested. Our electrophysiological findings together with our autoradiographic data provide strong evidence for the presence of specific and functional neurotensin receptors on astrocytes.