A detailed study of the immunoreactivity distribution of a novel pituitary protein, '7B2', within specific brain regions and in spinal cord revealed a complex network of neurons and fibers. Immunoreactive cell bodies were found exclusively in the hypothalamus. Stained fibers were more widely distributed and encompassed nearly 100 structures, including several limbic and autonomic nuclei. Biogenic amine centers such as substantia nigra, raphe nuclei and locus coeruleus have also contained immunoreactive 7B2. Subcellular fractionation using differential centrifugation and Percoll gradient revealed the preferential localization of this protein within granule-enriched fractions of rat neurointermediate lobe of the hypophysis. In agreement, ultrastructural studies revealed the presence of 7B2 within secretory-like granules in the median eminence and within synaptic vesicles of selected extrahypothalamic nuclei. Based upon migration on SDS-PAGE, the apparent molecular weight of the major form of 7B2 extracted from different brain regions was found to be 22,000, identical to that of the pituitary form.