The role of constitutive nitric oxide (NO) release in enteric neural pathways regulating ion transport was examined in guinea pig distal colon, in vitro and ex vivo. In in vitro studies, 43% of colonic preparations exhibited oscillations in baseline short-circuit current (Isc), which were reduced by tetrodotoxin (TTX). The NO chelator, hemoglobin (Hb), and neuronal NO synthase inhibitor, 7-nitroindazole (7-NI), significantly increased the baseline Isc in these tissues, which was reduced by TTX. In tissues without oscillations in baseline Isc, Hb reduced the Isc, while 7-NI had little effect. In all tissues, electrical field stimulation (EFS; 15 V/10 Hz) caused a biphasic increase in the Isc which was enhanced by both Hb and 7-NI. In the ex vivo studies, basal release of nitric oxide was significantly lower in colonic segments isolated from guinea pigs administered N omega-nitro-L-arginine methyl ester (L-NAME) i.p. compared to control tissues. Moreover, carbachol, caused a 10-fold increase in NO release in control tissues, but had no effect in tissues isolated from the L-NAME group. L-NAME increased tissue conductance and EFS-induced changes in Isc, which were reversed by L-arginine. However, carbachol-induced ion secretion was unaltered in the L-NAME group compared to control animals. The results suggest that, in guinea pig colon, constitutive enteric NO release tonically suppresses submucous neural activity and it is involved in the maintenance of basal epithelial chloride secretion and mucosal permeability. Hence, constitutive NO promotes a delicate balance between pro-absorptive and pro-secretory processes in guinea pig colon.