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The innervation of the muscularis mucosae of the canine large intestine was studied in vitro using superfusion and radioimmunological techniques. In the majority of preparations, electrical field stimulation (10 V, 200 microseconds, 10 Hz) elicited a biphasic neurogenic response which consisted of a contraction followed, after cessation of the stimulus, by relaxation. Electrical field stimulation released VIP-, substance P- and bombesin-like immunoreactivity. Release of these peptides and the biphasic response to nerve stimulation were blocked by tetrodotoxin and a 'calcium-free' solution. Several observations suggest that neuronally released substance P (or a closely related peptide) mediated the contraction by a direct action on the muscle. The contraction caused by substance P was tetrodotoxin insensitive. Desensitization to substance P abolished the excitatory response to nerve stimulation. The contraction elicited by nerve stimulation was blocked by substance P antiserum. Several observations suggest that bombesin or a closely related peptide caused contraction of the muscle by releasing substance P from intramural neurones. Bombesin caused an increase in substance P-like immunoreactivity in the superfusate which was blocked by tetrodotoxin, as was the contraction; substance P antibodies blocked the contractile response to bombesin. In addition, while the excitatory response to electrical nerve stimulation was blocked by substance P antiserum, there was still an increase in bombesin-like immunoreactivity in the superfusate. The data also suggest that VIP or a closely related peptide might have mediated the relaxation by a direct action on the muscle. The inhibitory response to nerve stimulation was mimicked by VIP and abolished by VIP antiserum.

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