This study was undertaken to find out the mechanism of non-agglutinable Vibrio cholerae heat-stable enterotoxin (NAG-ST)-induced calcium influx across the plasma membrane. Adriamycin, an inhibitor of IP3-specific 3-kinase, could not inhibit NAG-ST-induced calcium influx in rat intestinal epithelial cells, which suggested that inositol 1,3,4,5-tetrakisphosphate (IP4) had no role in NAG-ST-induced calcium influx. NAG-ST increased intracellular nitric oxide level of rat enterocytes as measured by a fluorimetric method using a fluoroprobe 4,5-diaminofluorescein-2-diacetate (DAF-2DA). N-Nitro-L-arginine, an inhibitor of nitric oxide synthase, inhibited NAG-ST-induced rise in nitric oxide level and also calcium influx. Inhibition of inositol trisphosphate (IP3)-mediated intracellular calcium mobilization by Dantrolene could also inhibit NAG-ST-induced rise in intracellular nitric oxide level. Moreover, inhibition of soluble guanylate cyclase by inhibitors (ODQ, LY83583) could inhibit the NAG-ST-induced rise in cyclic guanosine-3',5'-monophosphate (cGMP) level and calcium influx. From this study, it is evident that NAG-ST causes IP3-mediated calcium release from intracellular calcium store, which then stimulates nitric oxide production by activating nitric oxide synthase and the nitric oxide through cGMP activates calcium influx.