Anatomical and electrophysiological studies have demonstrated that enkephalinergic, noradrenergic, and serotonergic pathways projecting from the brain-stem to the dorsal horn inhibit nociceptive transmission at the spinal level. Previous attempts to delineate interactions between opioids, norepinephrine (NE), and serotonin (5-HT) in the production of spinal analgesia have produced conflicting results. The present study determined the effect of intrathecal (i.t.) pretreatment with opioid, NE, and 5-HT antagonists upon i.t. monoamine- and morphine-induced antinociception as assessed with the rat tail-flick model. Naloxone, at a dose which antagonized i.t. morphine analgesia, had no effect upon i.t. NE but inhibited i.t. 5-HT antinociception. Corynanthine or yohimbine (NE antagonists) reduced analgesia elicited by i.t. NE but not morphine, while pretreatment with methysergide or ketanserin (5-HT antagonists) attenuated both i.t. 5-HT- and morphine-induced antinociception. These results suggest that (1) an opioid link mediates spinal 5-HT but not NE antinociception, and (2) 5-HT but not NE participates in spinal morphine analgesia.