Activation of kappa-opioid receptors in the rostral ventral medulla has been reported to attenuate analgesia induced by activation of mu-opioid receptors in the periaqueductal gray matter. Previous studies have suggested that the cells associated with this effect might contain serotonin. In the present study, we investigated the relationship of the cloned kappa-opioid receptor to spinally projecting neurons immunoreactive for serotonin or GABA. This was done by employing two-color immunofluorescence in combination with retrograde tract-tracing using Fluoro-Gold. In the rostral ventral medulla, neurons triple-labeled for the cloned kappa-opioid receptor, serotonin and Fluoro-Gold were observed; neurons double-labeled for the cloned kappa-opioid receptor and serotonin, or single-labeled for the cloned kappa-opioid receptor or for serotonin were also observed. In addition, cloned kappa-opioid receptor immunoreactivity was expressed in some cell profiles immunoreactive for GABA. The expression of the cloned kappa-opioid receptor in the spinal cord dorsal horn was not associated with processes immunoreactive for serotonin. Our findings suggest that kappa-opioid receptors in the rostral ventral medulla are positioned to directly control the activity of at least some serotonergic neurons projecting to the dorsal spinal cord. Thus, it appears possible that the anti-analgesic action resulting from microinjection of kappa-opioid agonists into the rostral ventral medulla is mediated, at least in part, by these neurons.