The nature of cells in the hypothalamus that produce hypothalamic hypophysiotropic hormones remains unsettled. To investigate this problem electron microscopic immunocytochemistry was performed on thin sections of the median eminence of proestrous rats using antibodies to synthetic luteinizing hormone-releasing hormone (LHRH) and the peroxidase-antiperoxidase (PAP) technique. PAP complexes indicating the presence of LHRH were found over small, homogeneously dense neurosecretory granules 75-90 nm in diameter that occurred in clusters within neurons. Positive staining was more prevalent in the palisade zone in the anterior median eminence than at more posterior levels. LHRH positive granules were not obsereved in neuronal processes in the neurohemal contact zone, but appeared closer to the pericapillary space in the posterior median eminence than at more anterior levels. Occasional groups of LHRH positive granules were also found in the internal layer and hypendymal zone. No staining was observed in tanycytes or glial elements. These results support the hypothesis that hypothalamic hypophysiotropic hormones are produced by neurons and are stored in granules of homogeneous size and density in nerve processes located in the median eminence in the proximity of the hypophysial portal plexus.