Peripheral regulation of cardiovascular function is fundamentally influenced by central excitation and inhibition of sympathetic preganglionic neurons in thoracic spinal cord. This electron microscopy study investigated whether the γ-aminobutyric acid (GABA)-ergic and glycinergic inhibitory innervation of sympathetic preganglionic neurons arises from mutually exclusive afferent populations. Sympathetic preganglionic neurons were retrogradely labeled with cholera β subunit. GABAergic terminals were identified using strict quantitative statistical analyses as those boutons containing significantly elevated levels of GABA-like immunogold labeling (GABA+). Glycinergic terminals were classified as those boutons opposite postsynaptic gephyrin immunostaining containing background levels of GABA-like immunogold labeling (gephyrin+/GABA− association). Approximately 43% of the synaptic terminals that contacted sympathetic preganglionic somata and proximal dendrites and that were opposite gephyrin were GABA−; the remaining 57% were GABA+. Only two GABA+ boutons (4%) that synapsed on identified sympathetic preganglionic neuron (SPN) processes were not opposite gephyrin immunostaining (GABA+/gephyrin- association). GABA− /gephyrin+ associations were anticipated given prior anatomical, physiological, and pharmacological data. The observed nearly one-to-one correspondence between postsynaptic gephyrin immunoreactivity and GABA+ boutons was unexpected. Prior physiological and pharmacological experiments suggest that the postsynaptic effects of GABAergic inputs to sympathetic preganglionic neurons are mediated by activation of GABAA receptors. Those data, the present results, and other molecular, biochemical, and anatomical studies of gephyrin in the central nervous system (CNS) are consistent with two hypotheses: (1) Postsynaptic gephyrin is associated with GABAA receptors in the membranes of sympathetic preganglionic neurons, and (2) GABA+/gephyrin+ associations do not necessarily predict colocalization of GABA and glycine within single boutons synapsing on sympathetic preganglionic somata and dendrites. © 1995 Wiley-Liss, Inc.