The release of immunoreactive substance P (I-SP) from the myenteric plexus of the isolated guinea-pig small intestine and some of its characteristics have been investigated. Depolarizing stimuli, i.e. elevation of the extracellular K+ concentration or electrical field stimulation, increased the release of I-SP, the extent of the increase being dependent on the strength of the stimulus. Omission of Ca2+ from the bath medium prevented the stimulus-induced release of I-SP. Tetrodotoxin inhibited only the increase in I-SP release brought about by electrical stimulation but not that caused by elevated K+ concentrations. Gel exclusion chromatography on Sephadex G-25 showed that all the I-SP released by depolarization was co-eluted with authentic substance P. The methionine enkephalin analogue FK 33-824 significantly reduced the stimulus-induced release of I-SP, an effect that was prevented by the opiate antagonist naloxone. Naloxone alone significantly enhanced the stimulus-induced release of I-SP, which suggests that endogenously released opioid peptides also exert an inhibitory action on myenteric substance P-containing neurons. Putative excitatory neurotransmitters of the myenteric plexus, such as acetylcholine, bombesin, cholecystokinin octapeptide, and neurotensin, stimulated the release of I-SP in a tetrodotoxin-sensitive manner, whereas 5-hydroxytryptamine seemed ineffective. Capsaicin, known to release substance P from sensory neurons, also failed to alter the release of I-SP. The finding of a Ca2+-dependent release of I-SP caused by depolarizing stimuli further supports the concept that substance P is a neurotransmitter within the myenteric plexus. The activity of myenteric substance P-containing neurons appears to be controlled by a number of other putative enteric neurotransmitters.