Using a specific radioimmunoassay, we have found that immunoreactive (ir) alpha-neo-endorphin has a widespread and unique distribution in the rat brain and spinal cord. The highest concentration in brain is in the substantia nigra (1692.1 fmol/mg of protein). Very high concentrations of ir-alpha- neo-endorphin (greater than 500 fmol/mg of protein) are also found in the lateral preoptic nucleus, dentate gyrus, parabrachial nuclei, nucleus accumbens, globus pallidus, median eminence, and anterior hypothalamic nucleus. Relatively high concentrations of ir-alpha-neo-endorphin (250 to 500 fmol/mg of protein) are present in the bed nucleus of the stria terminalis, paraventricular nucleus, nucleus of the solitary tract, dorsomedial nucleus, central amygdaloid nucleus, periaqueductal gray matter, suprachiasmatic nucleus, periventricular nucleus, hippocampus, prepositus hypoglossal nucleus, arcuate nucleus, ventromedial nucleus, medial preoptic nucleus, zona incerta, dorsal premamillary nucleus, medial forebrain bundle (hypothalamic and preoptic), nucleus of the diagonal band, locus ceruleus, lateral septal nucleus, and nucleus ambiguus . Moderate levels (100 to 250 fmol/mg of protein) are found in the posterior hypothalamic nucleus, ventral premamillary nucleus, dorsal raphe nucleus, motor hypoglossal nucleus, caudate-putamen, periventricular thalamic nucleus, subcommissural organ, sensory trigeminal nucleus, perifornical nucleus, area postrema, supraoptic nucleus, cuneate nucleus, medial amygdaloid nucleus, and organum vasculosum laminae terminalis. Low concentrations of ir-alpha-neo-endorphin (less than 100 fmol/mg of protein) are found in many cortical structures, claustrum, thalamic nuclei, habenular nuclei, lateral geniculate body, red nucleus, superior and inferior colliculi, paramedian reticular nucleus, pontine nuclei, superior olive, vestibular nuclei, motor facial nucleus, gigantocellular reticular nucleus, and subfornical organ.(ABSTRACT TRUNCATED AT 250 WORDS)