Neurons in the rostral ventromedial medulla (RVM) are the major source of serotonergic projections to the dorsal horn. A large body of evidence implicates RVM serotonergic neurons in the modulation of spinal nociceptive transmission. Three physiological classes of RVM neurons, on, off, and neutral cells, are postulated to have different nociceptive modulatory effects on spinal nocifensor reflexes. This study was undertaken to determine which RVM cell class(es) contains 5- HT. In anesthetized rats, RVM neurons were identified by their responses to noxious cutaneous stimuli, intracellularly labeled, and processed for 5-HT immunocytochemistry. Labeled neurons were examined with epifluorescence and imaged using a confocal laser microscope. A total of 25 RVM neurons were intracellularly labeled. No off (n = 9) or on (n = 8) cells were serotonergic. Half of the neutral cells (4 of 8) demonstrated 5-HT immunoreactivity. These results call for a reevaluation of the mechanisms of RVM modulatory influence on spinal cord nociceptive transmission. The finding that some neutral cells are serotonergic strongly suggests that serotonergic neutral cells are involved in the modulation of spinal nociceptive transmission. Additionally, inhibition of spinal nociceptive transmission by off cells is unlikely to involve 5-HT release. Finally, since opioid administration does not alter the firing of RVM neutral cells, the results of the present study indicate that serotonergic RVM neurons do not directly mediate the effects of supraspinal opioids in the rat.