Supraspinal opioid analgesia is mediated in part by connections between the midbrain periaqueductal gray (PAG) and the ventral-medial medulla, including the nucleus raphe magnus (NRM) and nucleus reticularis gigantocellularis (NRGC). A serotonergic synapse appears to participate in this pathway since methysergide microinjected into the NRM-NRGC significantly reduced morphine analgesia elicited from the PAG. The present study evaluated the role of specific serotonin receptor subtypes by pretreating rats with microinjections of either the 5HT2 antagonist, ritanserin or the 5HT3 antagonist, ICS205930, into the NRM-NRGC and examining their effects upon morphine (2.5 micrograms) analgesia elicited from the PAG. Mesencephalic morphine analgesia was significantly reduced following pretreatment with both ritanserin (0.25-2.5 micrograms) on the tail-flick (81%) and jump (65%) tests and ICS205930 (0.25-5 micrograms) on the tail-flick (91%) and jump (63%) tests. Neither ritanserin nor ICS205930 altered basal nociceptive thresholds. Medullary placements ventral or lateral to the NRM/NRGC failed to support these antagonistic effects. These data indicate that ventro-medial medullary 5HT2 and 5HT3 serotonergic receptors modulate the transmission of opioid pain-inhibitory signals from the PAG.