An ultrastructural immunocytochemical study was undertaken to identify neuroactive substances contained in presynaptic boutons in the hypothalamic suprachiasmatic nucleus. Axonal boutons containing immunoreactive gamma-aminobutyrate, glutamate decarboxylase, neurophysin/vasopressin, gastrin releasing peptide/bombesin, somatostatin and serotonin were localized within the hypothalamic suprachiasmatic nucleus with pre-embedding peroxidase immunostaining. Synaptic contacts were found between boutons containing each of these substances and postsynaptic structures. While some variation in synaptic morphology existed, most of the immunoreactive contacts were of the symmetrical type. Previous work has indicated that neuroactive peptides may be found in highest concentrations in dense-core vesicles, to examine the subcellular localization of the amino acid inhibitory transmitter gamma-aminobutyrate, ultrastructural immunocytochemistry with pre-embedding peroxidase was compared with post-embedding immunocytochemistry with colloidal gold. Ultracryothin sections were also used for ultrastructural localization of gamma-aminobutyrate and glutamate decarboxylase immunoreactivity. Both gamma-aminobutyrate and glutamate decarboxylase immunoreactivity were found throughout the cytoplasm of immunoreactive boutons when pre-embedding peroxidase was used; with post-embedding colloidal gold immunostaining, label was found over areas containing small clear vesicles, and over mitochondria of immunoreactive axons. At the dilutions used in this study, strongly immunoreactive gamma-aminobutyrate dendrites, boutons forming asymmetrical synapses, and cell bodies were not found. Differences between pre-embedding and post-embedding immunostaining may be due to antigen and label diffusion caused by mild fixation and membrane damage necessary for antisera penetration during pre-embedding immunostaining. These results suggest that gamma-aminobutyrate, gastrin releasing peptide, somatostatin and vasopressin are contained in axons making contact with neurons of the suprachiasmatic nucleus, and may function as neurotransmitters here. Since all of these substances can also be localized in perikarya within the suprachiasmatic nucleus, there is a strong possibility that at least some of the axons containing immunoreactivity for each of these substances may be involved in local circuit interactions between neurons within the suprachiasmatic nucleus.