Recent evidence suggests that serotonin has pronociceptive actions in the spinal cord when it acts through 5-hydroxytryptamine (5-HT)(3) receptors. Cells and axon terminals which are concentrated in the superficial dorsal horn possess this receptor. We performed a series of immunocytochemical studies with an antibody raised against the 5-HT(3A) subunit in order to address the following questions: 1) Are axons that possess 5-HT(3) receptors excitatory? 2) Are 5-HT(3) receptors present on terminals of myelinated primary afferents? 3) What is the chemical nature of dorsal horn cells that possess 5-HT(3) receptors? 4) Do axons that possess 5-HT(3) receptors target lamina I projection cells? Approximately 45% of 5-HT(3A) immunoreactive boutons were immunoreactive for the vesicular glutamate transporter 2 and almost 80% formed synapse-like associations with GluR2 subunits of the AMPA receptor therefore it is principally glutamatergic axons that possess the receptor. Immunoreactivity was not present on myelinated primary afferent axons labeled with the B-subunit of cholera toxin or those containing the vesicular glutamate transporter 1. Calbindin (which is associated with excitatory interneurons) was found in 44% of 5-HT(3A) immunoreactive cells but other markers for inhibitory and excitatory cells were not present. Lamina I projection cells that possessed the neurokinin-1 receptor were associated with 5-HT(3A) axons but the density of contacts on individual neurons varied considerably. The results suggest that 5-HT(3) receptors are present principally on terminals of excitatory axons, and at least some of these originate from dorsal horn interneurons. The relationship between lamina I projection cells and axons possessing the 5-HT(3) receptor indicates that this receptor has an important role in regulation of ascending nociceptive information.