Pancreatitis is an inflammatory disease of pancreatic acinar cells whereby intracellular calcium ([Ca²⁺]_i) signaling and enzyme secretion is impaired. Increased oxidative stress has been suggested to mediate the associated cell injury. The present study tested the effects of the oxidant, hydrogen peroxide, on [Ca²⁺]_i signaling in rat pancreatic acinar cells by simultaneously imaging fura-2, to measure [Ca²⁺]_i, and dichlorofluorescein, to measure oxidative stress. Millimolar hydrogen peroxide increased cellular oxidative stress and irreversibly increased [Ca²⁺]_i, that was sensitive to antioxidants and removal of external Ca²⁺, and ultimately led to cell lysis. Responses were also abolished by pre-treatment with sarco/endoplasmic reticulum Ca²⁺-ATPase inhibitors, unless cells were pre-stimulated with cholecystokinin to promote mitochondrial Ca²⁺ uptake. This suggests that hydrogen peroxide promotes Ca²⁺ release from the endoplasmic reticulum, mitochondria and Ca²⁺ influx. Lower concentrations of hydrogen peroxide (10-100 µM) increased [Ca²⁺]_i and altered cholecystokinin-evoked [Ca²⁺]_i oscillations with marked heterogeneity, the extent of which was directly related to oxidative stress, suggesting differences in cellular antioxidant capacity. These changes in [Ca²⁺]_i also up-regulated the activity of the plasma membrane Ca²⁺-ATPase in a Ca²⁺-dependent manner, whereas higher concentrations (0.1 mM to 1 mM) inactivated the plasma membrane Ca²⁺-ATPase. This may be important in facilitating "Ca²⁺ overload" resulting in cell injury associated with pancreatitis.