Thealpha2C subclass of adrenergic receptor (alpha2C-AR) mediates some of the antinociceptive actions of norepinephrine in the spinal cord. Axon terminals, which possess this receptor, are concentrated in the superficial dorsal horn and originate from spinal interneurons. We performed a series of combined tract-tracing and immunocytochemical studies to determine whether alpha2C-AR-immunoreactive axons target projection neurons that possess the neurokinin-1 (NK-1) receptor because such cells are likely to transmit nociceptive information to the brain. Spinomedullary neurons were labeled by stereotaxic injection of the B-subunit of cholera toxin (CTb) into the caudal ventrolateral medulla of three anesthetized adult rats. After 3 d, the animals were anesthetized again and fixed by perfusion. Sections were cut from midlumbar segments and reacted with antibodies to reveal alpha2C-ARs, CTb, and NK-1 receptors. Retrogradely labeled neurons possessing the NK-1 receptor (n = 45) were examined with confocal microscopy to investigate their relationship with alpha2C-AR-immunoreactive axons. Numerous alpha2C-AR axons were apposed to cell bodies and proximal dendrites of cells in lamina I and also to distal dendrites that originate from labeled cell bodies in lamina III/IV. A combined confocal and electron microscopic method confirmed that these appositions were synaptic. Additional experiments showed that virtually all alpha2C-AR terminals in contact with labeled cells are also immunoreactive for the vesicular glutamate transporter 2 and therefore are glutamatergic. These data suggest that norepinephrine can modulate excitatory synaptic transmission from spinal interneurons to projection cells by acting at alpha2C-ARs. This could be one of the mechanisms that underlie the antinociceptive actions of norepinephrine.