The synaptic input from glutamate- or γaminobutyric acid (GABA)-immunoreactive nerve fibers to sympathetic preganglionic neurons projecting to the superior cervical ganglion was assessed using retrograde tracing with cholera toxin B subunit and post-embedding immunogold labelling. Half the boutons that synapsed on or directly contacted this group of neurons were immunoreactive for glutamate and half were immunoreactive for GABA. In previous studies, about 2/3 of the synapses on sympathoadrenal neurons were found to be glutamate-immunoreactive and about 1/3, GABA immunoreactive and these two amino acids were not co-localized. Thus, 100% of the boutons that synapse on sympathetic preganglionic neurons supplying the superior cervical ganglion or the adrenal medulla are likely to contain either glutamate or GABA. Anterograde tracing combined with amino acid immunocytochemistry indicated that at least some glutamate-containing and some GABA-containing nerve fibers in the intermediolateral cell column could come from the rostral ventral medulla.