The submucosal cholinergic and non-cholinergic neurons in the intestines have been shown to be involved in regulating epithelial transport functions, and particularly in stimulating Cl^- secretion. However, it is unclear whether electrogenic, amiloride-sensitive Na⁺ absorption in the colon is similarly controlled by these enteric neurons. This study investigates the role of the submucosal cholinergic neurons in regulating electrogenic Na⁺ absorption in the distal colon from the guinea pig treated with aldosterone. The amiloride (benzamil was also used)-sensitive short-circuit current (Isc) and ²²Na⁺ flux were measured in mucosal and mucosal-submucosal preparations mounted in Ussing chambers. In the mucosal preparation, the cholinergic agonist, carbachol (CCh), added to the serosal side inhibited the amiloride-sensitive Isc and amiloride-sensitive ²²Na⁺ absorption. The inhibitory effect of CCh was observed at ~0.1 µM, and the maximum inhibition of approximately 70% was attained at ~30 µM, the IC50 value being approximately 1 µM. The CCh-induced inhibition of amiloride-sensitive Isc was almost totally abolished by 10 µM atropine. Treatment of the tissue with the Ca²⁺-ionophore, ionomycin, markedly reduced the amiloride-sensitive Isc, but a subsequent addition of CCh further decreased it. In addition, CCh still had an inhibitory effect, although significantly attenuated, after the tissue had been incubated with a low-Ca²⁺ solution containing ionomycin and BAPTA-AM. Applying electrical field stimulation to the submucosal neurons in the mucosal-submucosal preparation resulted in the inhibition of amiloride-sensitive Isc, approximately one third of this inhibition being atropine-sensitive. The cholinesterase inhibitor, physostigmine, inhibited the amiloride-sensitive Isc, this effect being abolished by atropine. In conclusion, submucosal cholinergic and non-cholinergic neurons were involved in inhibiting electrogenic Na⁺ absorption in the colon. This inhibition by cholinergic neurons was mediated by muscarinic receptor activation.