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1. Acetylcholine increases the potential difference across rat proximal colon both in vivo and in vitro.

2. There is a sigmoid relationship between the change in potential difference and the logarithm of the dose of acetylcholine. The dose—response curve is shifted to the left by neostigmine and to the right by atropine, suggesting that the action of acetylcholine is mediated by a muscarinic type of receptor.

3. The dose-response curve for acetylcholine in vivo is not altered by the ganglion-blocking agents hexamethonium and pentolinium, suggesting a direct effect of this transmitter on the colon.

4. Acetylcholine causes an increase in potential difference, a small decrease in resistance and hence a rise in the current generated by both normal and stripped everted sacs of rat colon.

5. In the absence of sodium, the calculated current change produced by acetylcholine is reduced, and the removal of chloride has a similar inhibitory effect. The absence of bicarbonate does not significantly affect the response.

6. Acetylcholine virtually abolished net sodium movement and induced net chloride secretion and these changes accounted for the increased short-circuit current.

7. Acetylcholine had no effect on oxygen consumption by rings of colon.

8. Tracts staining for acetylcholinesterase were observed running from the submucous plexus towards the mucosal epithelium.

9. This study shows that acetylcholine can influence ion movement by rat colonic mucosa and suggests that the autonomic nervous system might be involved in the regulation of transport mechanisms in this tissue.