In an attempt to explore the functioning of nitric oxide (NO) in pancreatic exocrine cells, we have recently obtained several lines of circumstantial evidence indicating that one of molecular targets of NO is phospholipase C (PLC), the activation of which leads to an increase in the cytosolic Ca2+ concentration ([Ca2+]i) via inositol 1, 4, 5-trisphosphate, IP3. However, whether IP3 is actually produced by NO has not yet been substantiated. The present study was therefore designed to directly measure the intracellular IP3, concentration ([IP3]i) for better understanding of the underlying mechanisms with the help of pharmacological tools. [IP3]i was measured using a fluorescence polarization technique (HitHunter). We obtained the following results: 1) varying concentrations of an NO donor, sodium nitroprusside (SNP), elevated [IP3]i, 2) this elevation was completely inhibited in the presence of the soluble guanylyl cyclase (sGC) inhibitor, 1H-[1, 2, 4] oxadiazolo [4, 3-a] quinoxalin-1-one (ODQ), 3) varying concentrations of the cGMP analogue, 8-Br-cGMP, also increased [IP3]i, 4) the cGMP analogue-induced IP3 production was abolished by pretreatment with either a PLC inhibitor, U73122, or a G-protein inhibitor, GP2A, and 5) KT5823, a potent and highly selective inhibitor of cGMP-dependent protein kinase G (PKG), also abolished the IP3 production induced by 8-Br-cGMP. These results suggest that the NO-induced [Ca2+]i increase is triggered by an increase in [IP3]i located downstream from intracellular cGMP elevation. In this intracellular pathway, each sGC, cGMP-dependent PKG, G-protein and PLC were suggested to be involved. The present work provides new insights into the intracellular signaling accelerated by NO. NO triggers a [Ca2+]I increase via cGMP and IP3 in pancreatic acinar cells.