Glutamate immunoreactivity was examined in the sympathetic intermediolateral nucleus (IML) of the thoracic spinal cord using an antibody (Hepler et al., J. Histochem. Cytochem., 36 (1988) 13-22) to hemocyanin-conjugated L-glutamate and the peroxidase-antiperoxidase (PAP) technique. Glutamate labeling was seen in punctate varicosities throughout the IML, in IML neurons and their dendritic processes extending into the lateral funiculus, and diffusely in the IML neuropil. Glutamate labeling was unaffected by preabsorption of the antibody with a conjugate of L-aspartate (100 micrograms/ml) to bovine serum albumin (BSA), but was abolished by similar treatment with a conjugate of L-glutamate (1.7 microgram/ml) to BSA. Glutamate immunoreactivity in the IML was eliminated caudal to T3 spinal transection, while that in the dorsal horn was preserved. Ultrastructural examination of the IML revealed that glutamate was localized within round clear vesicles in axon terminals ranging in diameter from 0.6 to 2.5 microns, and containing several mitochondria, but no dense-core vesicles. Glutamate-containing terminals made primarily asymmetric synapses on small dendrites of IML neurons. These synapses were usually enveloped by processes of astrocytic glia which could also contain glutamate immunoreactivity. The findings provide an anatomic substrate for glutamatergic excitation of sympathetic nerve discharge through a descending spinal pathway terminating in the IML. These data support the hypothesis that sympathoexcitation elicited by stimulation of the rostral ventrolateral medulla may be mediated by a direct glutamatergic pathway to the IML.