Introduction: L-Asparaginase is an essential element of combination chemotherapy in the treatment of acute lymphoblastic leukaemia (ALL). However, asparaginase-induced acute pancreatitis is the most common reason (7-18%) for stopping the relatively successful treatment of childhood ALL (Raja et al, 2012). Acute pancreatitis is a dangerous human disease characterized by premature activation of digestive enzymes inside the pancreatic acinar cells that leads to high levels of necrosis, digestion of the pancreatic tissue and its surroundings with no specific therapy (Reference). The aim of the present study was to investigate the molecular mechanisms and the signaling pathways of cellular injury in pancreatic acinar cells induced by L-asparaginase. Methods: Pancreatic acinar cells were isolated as previously described (Ferdek et al 2012) according to the Animal Scientific Procedures Act, 1986. Cytosolic Ca2+ ([Ca2+]i) measurements were performed with Fura-2 or Fluo-4 imaging. Mitochondrial calcium ([Ca2+]m) and mitochondrial membrane potential were measured using Rhod-2 AM and TMRM, respectively. Magnesium green (MgGreen) was employed to assess cellular ATP depletion, whereas necrosis was assessed with propidium iodide staining. Results: We found that L-asparaginase (starting from 0.1 U/ml) induces small [Ca2+]I responses in pancreatic acinar cells, while higher doses L-asparaginase (200 U/ml) leads to large and prolonged (plateau) responses of [Ca2+]I. L-asparaginase also significantly increased calcium entry and substantially reduced calcium extrusion. L-asparaginase treatment has also resulted in significant reduction of cellular ATP levels. In addition, the results showed that L-asparaginase induce substantial increase in mitochondrial calcium and decrease in mitochondrial membrane potential. Calmodulin activators CALP-3, CALPm (Gerasimenko et al, 2014) and inhibitor of calcium entry GSK-7975A (Gerasimenko et al, 2013) have protected against reduction of cellular ATP levels induced by L-asparaginase and subsequent necrosis. Conclusions: These data suggest that L-asparaginase affects pancreatic acinar cell fate via modulating calcium signalling and mitochondrial functions. Calmodulin activators are highly effective against calcium overload, cellular ATP depletion and necrosis induced by L-asparaginase. Thus, calmodulin activators may have important therapeutic implications for improving prognosis, prevention of acute pancreatitis and improvement of childhood cancer treatments.