The rostral ventromedial medulla (RVM) forms part of a descending pathway that modulates nociceptive neurotransmission at the level of the spinal cord dorsal horn. However, the involvement of descending RVM systems in opioid analgesia are a matter of some debate. In the present study, patch-clamp recordings of RVM neurons were made from rats that had received retrograde tracer injections into the spinal cord. More than 90% of identified spinally projecting RVM neurons responded to opioid agonists. Of these neurons, 53% responded only to the μ-opioid agonist d-Ala2, N-Me-Phe4, Gly-ol5 enkephalin, 14% responded only to the κ-opioid agonist U-69593, and another group responded to both μ and κ opioids (23%). In unidentified RVM neurons, a larger proportion of neurons responded only to μ opioids (75%), with smaller proportions of κ- (4%) and μ/κ-opioid (13%) responders. These RVM slices were then immunostained for tryptophan hydroxylase (TPH), a marker of serotonergic neurons. Forty-percent of spinally projecting neurons and 11% of unidentified neurons were TPH positive. Of the TPH-positive spinally projecting neurons, there were similar proportions of μ- (33%), κ- (25%), and μ/κ-opioid (33%) responders. Most of the TPH-negative spinally projecting neurons were μ-opioid responders (67%). These findings indicate that functional opioid receptor subtypes exist on spinally projecting serotonergic and nonserotonergic RVM neurons. The proportions of μ- and κ-opioid receptors expressed differ between serotonergic and nonserotonergic neurons and between retrogradely labeled and unlabeled RVM neurons. We conclude that important roles exist for both serotonergic and nonserotonergic RVM neurons in the mediation of opioid effects.