Neurons likely to use endothelin as a neurotransmitter/neurohormone were mapped in the rat brain using polyclonal antibodies directed against endothelin-converting enzyme-1, endothelin-1, and endothelin-3. Anti-endothelin-converting enzyme-1 antibodies produced the most robust staining, permitting the best visualization of the distribution and morphology of neurons. Labeled neurons were found in the dorsal thalamic nuclei and reticular thalamic nuclei, medial preoptic area, pontine nucleus, and locus coeruleus. Localization of endothelin-converting enzyme-like immunoreactivity in the locus coeruleus and in the reticular nucleus of the thalamus suggests that endothelin is co-localized with norepinephrine and GABA, respectively. Additionally, endothelin-converting enzyme-like immunoreactivity was found in the globus pallidus, septal nuclei, and in both the vertical and horizontal limbs of the nucleus of the diagonal band of Broca, and the ventrolateral area of the caudate-putamen. Strong endothelin-converting enzyme-like immunoreactivity was found in a continuous band of pyramidal neurons throughout the neocortex primarily in layer V, extending into the cingulate gyrus and piriform cortex. Motor nuclei, including oculomotor, facial, and trigeminal nuclei, were also endothelin-converting enzyme-immunoreactive. In the cerebellum, Purkinje cells were stained. Non-neuronal cells such as oligodendroglia, microglia, and astrocytes generally were not endothelin-converting enzyme-immunoreactive, although astrocytes were rarely stained. Endothelin-converting enzyme-, endothelin-1-, and endothelin-3-like immunoreactivities were generally found co-existing in given nuclei. The diversity of neurons immunostained for endothelin suggests multiple roles of endothelin in the CNS.