Endothelin binding sites in rat brain were mapped by quantitative in vitro autoradiography employing [125I]endothelin-1 as radioligand. [125I]Endothelin-1 bound with high affinity and specificity to rat cerebellar sections and was potently displaced by unlabelled endothelins (endothelin-1 greater than endothelin-2 = endothelin-3) and sarafotoxin 6B. The highest densities of endothelin binding sites were found in the cerebellum (especially Purkinje cell layer), choroid plexus and median eminence. High densities were found in the supraoptic and paraventricular hypothalamic nuclei, anterior hypothalamic area, ventromedial hypothalamic nucleus, mammillary nuclei and glomerular layer of olfactory bulb. Moderate densities were found in many thalamic nuclei, the pretectal region, interpeduncular nucleus, suprachiasmatic nucleus, raphe nuclei, tegmental nuclei, olfactory ventricle, red nucleus, subthalamic nucleus, central gray, reticular nuclei, vestibular nuclei, oculomotor and trochlear nuclei, hypoglossal nucleus, motor trigeminal nucleus, nucleus of the trapezoid body and lateral cerebellar nucleus. Low but detectable densities of endothelin binding sites were found in medial geniculate nucleus, fields of Ammon's horn, caudate-putamen, globus pallidus, entopeduncular nucleus, substantia nigra, anterior commissure, internal capsule, anterior pituitary, median preoptic nucleus, septohypothalamic nucleus, superior colliculus and area postrema. These patterns were completely abolished by 1 microM unlabelled endothelin-1, -2 and -3 and sarafotoxin S6B. Brain endothelin binding sites show high affinity for endothelin-1, -2 and -3 and sarafotoxin 6B with highest affinity for endothelin-1. Endothelin binding sites show a non-vascular pattern of distribution in the brain, suggesting that the peptide may have widespread functions as a modulator of neuronal function.