To determine the localization in rat brain and spinal cord of individual neurons that contain the messenger RNA coding for the opioid peptide precursor preproenkephalin, we performed in situ hybridization with a tritiated cDNA probe complementary to a protion of preproenkephalin mRNA. We observed autoradiographic signal over the cytoplasm of neurons of many regions of the central nervous system. Several types of controls indicated specificity of the labeling. Neurons containing preproenkephalin mRNA were found in the piriform cortex, ventral tenia tecta, several regions of the neocortex, nucleus accumbens, olfactory tubercle, caudate-putamen, lateral septum, bed nucleus of the stria terminalis, diagonal band of Broca, preoptic area, amygdala (especially central nucleus, with fewer labeled neurons in all other nuclei), hippocampal formation, anterior hypothalamic nucleus, perifornical region, lateral hypothalamus, paraventricular nucleus, dorsomedial and ventromedial hypothalamic nuclei, arcuate nucleus, dorsal and ventral premamillary nuclei, medial mamillary nucleus, lateral geniculate nucleus, zona incerta, periaqueductal gray, midbrain reticular formation, ventral tegmental area of Tsai, inferior colliculus, dorsal and ventral tegmental nuclei of Gudden, dorsal and ventral parabrachial nuclei, pontine and medullary reticular formation, several portions of the raphe nuclei, nucleus of the solitary tract, nucleus of the spinal trigeminal tract (especially substantia gelatinosa), ventral and dorsal cochlear nuclei, medial and spinal vestibular nuclei, cuneate and external cuneate nuclei, gracile nucleus, superior olive, nucleus of the trapezoid body, some deep cerebellar nuclei, Golgi neurons in the cerebellum, and most laminae of the spinal cord. In most of these brain regions, the present results indicate that many more neurons contain preproenkephalin mRNA than have been appreciated previously on the basis of immunocytochemistry.