L-Asparaginase is an essential element in the successful treatment of Acute Lymphoblastic Leukaemia (ALL), the most common type of cancer to affect children. Unfortunately, Asparaginase treatment results in acute pancreatitis (AAP) in about 5 – 10% of cases forcing to discontinue Asparaginase treatment. Our main approach for studies of physiological/pathophysiological signalling in pancreatic cells was the monitoring of Ca2+ concentrations in the cytoplasm and organelles. Pancreatic acinar cells were freshly isolated from mice. Animals were sacrificed according to the Animal Scientific Procedures Act, 1986 and approved by the Ethical Review Committee of Cardiff University. After dissection the pancreas was digested using collagenase-containing solution (200 U/ml, Worthington, UK). For measurements of [Ca2+]i, isolated pancreatic acinar cells were loaded with Fluo-4-AM (5 μM). Necrotic cell death was assessed with propidium iodide uptake. Data are presented as mean ± SEM, statistical significance and P values calculated using t-test or ANOVA with P<0.05 considered significant. Monitoring Ca2+ concentrations in the cytoplasm, we have shown for the first time that intracellular Ca2+ release followed by Ca2+ entry and resulting in [Ca2+]I plateau is the main type of Ca2+ response to Asparaginase. The primary intracellular Ca2+ release was largely dependent on IP3 and NAADP signalling mechanisms. The IPR3 blocker caffeine (20 mM) markedly inhibited the Asparaginase-induced [Ca2+]i elevations in the absence of external Ca2+ (n=8). The PLC inhibitor has blocked the Asparaginase-induced [Ca2+]i elevation (n=11). ACh (1 μM) was applied at the end of each experiment but did not elicit any change in [Ca2+]i. Ryanodine (100 μM) markedly inhibited the Asparaginase-induced Ca2+ signals (n=13). Ned-19 (100 μM) has also prevented the Asparaginase-induced [Ca2+]i elevation (n=8). The Asparaginase-induced Ca2+ signals were practically eliminated by the protease-activated receptor 2 (PAR2) inhibitor FSLLLRY-NH2 (10μM) (n=32). The toxic Ca2+ signals caused significant necrosis (17.4%, n>250 in each independent series of experiments). Inhibition of calcium signals as well as PAR2, or pyruvate supplementation significantly blocked Asparaginase-induced necrosis. Understanding AAP pathogenesis could lead to development of effective therapies for this complication, potentially reducing toxicity and allowing re-exposure to continued treatment with Asparaginase.