Porcine distal colon epithelium was mounted in Ussing chambers and bathed with porcine Ringer's solution. The effects of vasoactive intestinal polypeptide, norepinephrine, and carbamylcholine on Na and Cl fluxes and transepithelial electrical parameters were determined after their serosal administration. Vasoactive intestinal peptide increased the Cl-dependent component of the short-circuit current with a half-maximal effect at 115 nmol/L. Transepithelial Na and Cl flux studies demonstrated that the increase in current was caused by stimulation of Cl secretion. Norepinephrine also stimulated Cl secretion and increased the serosal-to-mucosal Na flux, producing a half-maximal effect at 1.6 μmol/L. Selective blockade of α1 adrenoceptors by 0.5 μmol/L prazosin produced an eightfold decrease in norepinephrine potency. Carbamylcholine produced a significant increase in Cl secretion and decreased absorption of both Na and Cl with a concentration of 10 μmol/L producing a half-maximal effect. The muscarinic cholinoceptor blocker atropine (0.1 μmol/L) produced a 22-fold decrease in carbamylcholine potency. The effects of all three transmitter substances were unaffected after pretreatment of tissues with the neuronal conduction-blocker tetrodotoxin or an inhibitor of arachidonic acid metabolism. These results indicate that (a) vasoactive intestinal polypeptide stimulates Cl secretion without affecting Na absorption; (b) norepinephrine acting through α1 adrenoceptors stimulates net Cl secretion and activates a serosal-to-mucosal Na transport mechanism; and (c) carbamylcholine acting through muscarinic receptors stimulates Cl secretion and inhibits Na and Cl absorption.