Segments of guinea pig distal colon, stripped of the external muscle layers, were set up in flux chambers for measurement of short-circuit current (Isc) indicative of active, electrogenic ion transport. During neural blockade with tetrodotoxin, the nitric oxide scavenger, hemoglobin, and the nitric oxide synthase inhibitor, N omega-nitro-L-arginine (L-NNA), reduced Isc. The reduction in Isc in response to hemoglobin was reversed by L-arginine and blockers of chloride secretion, including bumetanide and diphenylamine-2-carboxylic acid, but not by the potassium channel blockers, barium and tetraethylammonium, nor by amiloride, an epithelial sodium channel blocker. The hemoglobin-induced reduction in Isc was not affected by blockade of prostaglandin synthesis with piroxicam. During neural blockade, the nitric oxide donors, sodium nitroprusside and NONOate, increased Isc which was abolished by piroxicam. Endothelin-1 (ET-1) also evoked an increase in Isc that was unaffected by amiloride and was inhibitable by bumetanide, chloride-free solutions, tetrodotoxin, piroxicam, and the ETA receptor antagonist, BQ123. The ETB receptor agonist, [Ala1,3,11,15]-endothelin-1, had no appreciable effect on Isc. Hemoglobin and L-NNA enhanced the ET-1-induced Isc response by about twofold without affecting prostaglandin E2 release or its secretory response. The results suggest that endogenous nitric oxide stimulates a low level of chloride secretion that is independent of prostaglandins, unlike nitric oxide donors which increase chloride secretion by releasing prostaglandins. In addition, endogenous nitric oxide suppresses ET-1-evoked chloride secretion by mechanisms that are unrelated to the release of prostaglandin E2 or its ability to stimulate epithelial cells. Endogenous nitric oxide may play an important role in modulating chloride secretion during ischemic challenge when endothelin levels are high.