1. Strips of longitudinal muscle can be obtained from guinea-pig ileum either retaining or free from Auerbach's plexus.2. The denervated strip is unresponsive to electrical stimulation by brief shocks, whether given singly or in trains; it also fails to respond to nicotine or dimethylphenylpiperazinium iodide (DMPP), and eserine causes no spasm.3. Denervated strips neither contain detectable acetylcholine (< 0.4 ng/mg), nor release it spontaneously (< 5 pg/mg/min) or in response to stimulation (< 31 pg/mg/min). The acetylcholine metabolism of the innervated strip is therefore that of the adherent Auerbach's plexus. Innervated strips had a mean acetylcholine content of 28 ng/mg, a mean resting output of 94 pg/mg/min and an output in response to stimulation at 10 c/s of 700-1200 pg/mg/min.4. By comparing the responses of innervated and denervated strips it was concluded that arecoline, methylfurmethide, alpha,beta-ethylal-gamma-tri-methylammonium propanediol iodide (2268 F), muscarine, histamine, tremorine, oxytocin, and substance P, like acetylcholine, act primarily on the smooth muscle directly; and that angiotensin, barium, potassium, m-bromophenyl choline ether and 5-hydroxytryptamine have a progressively increasing proportionate effect on the nerve plexus. Nicotine and DMPP were inactive in the absence of the plexus.5. The longitudinal muscle with its accompanying plexus contains about one quarter of the acetylcholine of the whole ileum, and is responsible for about one fifth of the output to electrical stimulation.6. The volley output of acetylcholine by the innervated strip declines sharply as rate of stimulation increases. Output of acetylcholine was reduced by morphine and by cocaine, particularly when resting or when stimulated at low rates.7. Acetylcholine output by whole ileum from guinea-pig declines in the absence of glucose, but is insulin-independent. Output by strips of ileum from rats made diabetic with alloxan was similar to that from normal rats.8. The similarity in properties of acetylcholine output from innervated strips, where it must come from nervous tissue, to that from whole ileum, and the insulin-independence of output from whole ileum suggest that the whole of the acetylcholine output of intestine is nervous in origin.9. Comparison of the acetylcholine metabolism of the innervated strip with that of the superior cervical ganglion suggests that the typical features of the former (high resting output, high volley output at low rates, low minute output at high rates of stimulation, and sensitivity to morphine) may be linked with the absence of specialized neuro-effector junctions and represent a relatively primitive transmission process.