In the normal colon acetylcholine elicits a luminally-directed Cl^- efflux from enterocytes via activation of muscarinic receptors. In contrast, in the murine model of dextran sodium sulfate (DSS) colitis, an inhibitory cholinergic ion transport event due to nicotinic receptor activation has been identified. The absence of nicotinic receptors on enteric epithelia and the ability of nitric oxide (NO) to modulate ion transport led us to hypothesize that NO mediated the cholinergic nicotinic-induced changes in ion transport. Mid-portions of colon from control and DSS-treated mice were examined for inducible NO synthase (iNOS) expression by RT-PCR and immunofluorescence or mounted in Ussing chambers for assessment of cholinergic-evoked changes in ion transport (i.e. short-circuit current) ± pretreatment with pharmacological inhibitors of NO production. iNOS mRNA and protein levels were increased throughout the tissue from DSS-treated mice and notably in the myenteric plexus, where the majority of the iNOS immunoreactivity co-localized with the enteric glial cell marker, glial fibrillary acidic protein. The drop in I_SC evoked by the cholinomimetic, carbachol, in tissue from DSS-treated mice was prevented by selective inhibitors of iNOS activity (L-NIL, 1400w), a NO scavenger (cPTIO), or by removal of the myenteric plexus. Thus, in this model of colitis a "switch" occurs from muscarinic to nicotinic receptor-dominated control of cholinergic ion transport. The data indicate a novel pathway involving activation of nicotinic receptors on myenteric neurons resulting in the release of NO from neurons or enteric glia, and ultimately a dampening of stimulated epithelial Cl^- secretion that would reduce secretory diarrhea.