Cholinergic agonists inhibit sodium and chloride absorption in the intestine and often produce secretion. To determine the mechanism of cholinergic regulation of intestinal electrolyte transport, the effects of bethanechol on ion transport were studied in the rat colon in vitro. The addition of 1 mM bethanechol produced an initially large but short-lived increase in short-circuit current (Isc) (171 +/- 19 microA/cm2). Bethanechol decreased net sodium and net chloride absorption (2.5 +/- 0.5 and 2.9 +/- 0.9 mueq . h-1 . cm-2, respectively) and increased Isc (0.8 +/- 0.3 mueq . h-1 . cm-2) during the steady-state period. All these effects were inhibited by 1 microM atropine, which alone had no effect on ion transport. The removal of either sodium or chloride also inhibited the effect of bethanechol. cAMP content did not increase in isolated enterocytes incubated with bethanechol; however, calcium removal from the serosal bathing solution inhibited the bethanechol-induced changes in ion transport. These results indicate that cholinergic muscarinic agonists alter sodium and chloride transport in the colon by inhibiting coupled NaCl absorption by a calcium-dependent, non-cAMP-mediated process.