Uptake of [3H]choline and the subsequent synthesis and release of [3H]acetylcholine was studied in a preparation of rat colonic mucosa in vitro. [3H]Choline uptake was inhibited by hemicholinium-3. Release of synthesized [3H]acetylcholine was increased significantly over base line by 50 mM K+ and 100 microM veratridine. K+-evoked release was inhibited by 83% in tissue preincubated (during [3H]choline uptake) in Na+-free media. The effect of K+ was also blocked in Ca++-free media by 15 mM Mg++ and 0.1 mM verapamil. Release from the longitudinal and circular muscle of the colon evoked by K+ and veratridine was greater than release from the mucosa. Tetrodotoxin (0.2 microM) did not affect K+-induced release of [3H] acetylcholine but significantly blocked veratridine-induced release from the mucosal (by 63%) and muscle (by 68%) preparations. The data confirm that functional cholinergic nerve endings exist in close proximity to colonic epithelial cells.