Bacterial toxins such as cholera toxin induce diarrhea by both direct epithelial cell generation of cyclic nucleotides as well as stimulation of the enteric nervous system (ENS). Agonists of the extracellular calcium-sensing receptor (CaSR) can reduce toxin-stimulated fluid secretion in ENS-absent colonic epithelial crypts by increasing phosphodiesterase-dependent cyclic-nucleotide degradation. Here we show that the CaSR is also highly expressed in tetrodotoxin (TTX)-sensitive neurons comprising the ENS, suggesting that CaSR agonists might also function through neuronal pathways. To test this hypothesis, rat colon segments containing intact ENS were isolated and mounted on Ussing chambers. Basal and cyclic nucleotide-stimulated electrolyte secretions were monitored by measuring changes in short-circuit current (I(sc)). CaSR was activated by R-568 and its effects were compared in the presence and absence of TTX. Consistent with active regulation of anion secretion by the ENS, a significant proportion of I(sc) in the proximal and distal colon was inhibited by serosal TTX, both at basal and under cyclic AMP-stimulated conditions. In the absence of TTX, activation of CaSR with R-568 significantly reduced basal I(sc) and cyclic AMP-stimulated I(sc); it also completely reversed the cAMP-stimulated secretory responses if the drug was applied after the forskolin stimulation. Such inhibitory effects of R-568 were either absent or significantly reduced when serosal TTX was present, suggesting that this agonist exerts its antisecretory effect on the intestine by inhibiting ENS. The present results suggest a new model for regulating intestinal fluid transport in which neuronal and nonneuronal secretagogue actions are modulated by the inhibitory effects of CaSR on the ENS. The ability of a CaSR agonist to reduce secretagogue-stimulated Cl(-) secretion might provide a new therapeutic approach for secretory and other ENS-mediated diarrheal conditions.