Electrical field stimulation was used to activate enteric neurons in the guinea pig ileum in order to determine the involvement of intramural cholinergic motor neurons in the control of mucosal sodium and chloride transport. Rectangular, bipolar stimulus pulses evoked a tetrodotoxin-sensitive biphasic increase in short-circuit current (Isc) that was due to an increase in active chloride secretion without any effect on active sodium absorption. During activation of enteric neurons, atropine, in concentrations of 5 X 10(-8) M or higher, reversibly abolished the rapid phase of the Isc and reduced the maximum sustained response to 40-50% of control values. Atropine reduced chloride secretion evoked by electrical stimulation and had no effect on sodium fluxes. Basal rates of sodium and chloride transport and tissue conductances were not altered by atropine. When muscarinic receptors were maximally stimulated with 10(-5) M carbachol, activation of enteric nerves increased Isc to 46% of control values. Bethanechol and eserine increased base-line Isc, and this effect was blocked by atropine but not by tetrodotoxin. The increase in Isc evoked by bethanechol was due to an increase in active chloride secretion. These results show that acetylcholine released by activation of enteric cholinergic motor neurons acts as muscarinic receptors on enterocytes to stimulate chloride secretion.