The relationship between neuropeptides and serotonergic neurones was investigated in rat spinal cord, in vivo, using a subarachnoidal perfusion technique. The 5-hydroxytryptamine (5-HT) release from the spinal cord could be evoked by substance P and peripheral pain stimulation (tail pinch), but not by methionine-enkephalin (met-EK). The substance P-evoked 5-HT release was slightly potentiated by met-EK. The tail pinch-evoked 5-HT release was inhibited by met-EK and baclofen, but potentiated by naloxone. These findings may infer a possible mechanism by which neuroactive peptides regulate 5-HT release from serotonergic neurones in the spinal cord.