Submucosal cholinergic and noncholinergic neurons in intestines have been shown to be involved in regulating epithelial transport functions, particularly stimulating Cl secretion. This study investigates the role of submucosal cholinergic neurons in regulating electrogenic Na⁺ absorption in distal colon. Amiloride-sensitive short-circuit current (I_sc) and ²²Na⁺ flux were measured in mucosal and mucosal-submucosal preparations mounted in Ussing chambers. In the mucosal preparation, carbachol (CCh) added to the serosal side inhibited amiloride-sensitive I_sc and amiloride-sensitive ²²Na⁺ absorption. The inhibitory effect of CCh was observed at ~0.1 µM, and maximum inhibition of ~70% was attained at ~30 µM (IC₅₀ = ~1 µM). CCh-induced inhibition of amiloride-sensitive I_sc was almost totally abolished by 10 µM atropine. Treatment of the tissue with ionomycin markedly reduced amiloride-sensitive I_sc, but a subsequent addition of CCh further decreased it. Also, 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 submucosal neurons in the mucosal-submucosal preparation resulted in inhibition of amiloride-sensitive I_sc, ~33% of this inhibition being atropine sensitive. Physostigmine inhibited amiloride-sensitive I_sc, this effect being abolished by atropine. In conclusion, submucosal cholinergic and noncholinergic neurons were involved in inhibiting electrogenic Na⁺ absorption in colon. This inhibition by cholinergic neurons was mediated by muscarinic receptor activation.