The relationship between cytosolic calcium [Ca2+]c, reactive oxygen species (ROS) and mitochondrial function plays an important role in cellular physiology and pathology, yet is currently poorly defined. We have previously shown that oxidant stress promotes apoptotic pancreatic acinar cell death (Criddle et al., 2006) and have now investigated the influence of [Ca2+]c on ROS generation, mitochondrial function and cell fate in isolated murine pancreatic acinar cells. Isolated murine pancreatic acinar cells were exposed to taurolithocholic acid sulphate (TLC-S; 500µM) or the quinone oxidant menadione (MEN; 30µM) and examined by confocal microscopy to detect ROS (CM-H2DCFDA), [Ca2+]c (Fluo-4), NAD(P)H (autofluorescence, a measure of mitochondrial metabolism), mitochondrial Ca2+ ([Ca2+]mt; Rhod-2), and apoptosis (caspase activaton: R110-aspartic acid amide). The antioxidant N-acetyl-L-cysteine (NAC; 10mM) was applied as a ROS scavenger and BAPTA-AM (25µM) as a Ca2+ chelator. TLC-S caused a large, sustained rise of [Ca2+]c above basal levels and a NAC-sensitive generation of ROS (n=16). Mitochondrial function was inhibited by application of TLC-S; a large, sustained increase of [Ca2+]mt was observed with a concomitant decrease of NAD(P)H( n=18). Both the sustained rises of [Ca2+]c and ROS were abolished by BAPTA-AM pre-treatment, indicating the Ca2+-dependency of TLC-S-induced ROS generation (n=14). In contrast, MEN-induced elevation of ROS was unaffected by BAPTA pre-treatment, but blocked by NAC (n=16), in accord with a Ca2+-independent redox cycle mechanism. When cells were treated with TLC-S or MEN for 30 minutes, apoptotic cell death was increased 3.2-fold and 5.5 fold from control levels (n=1132, 632), respectively, in a manner that was abolished by treatment with NAC, indicating a ROS-dependency of the actions of both compounds. Reactive oxygen species, generated by distinct Ca2+-dependent and independent mechanisms, are important mediators of apoptosis in the pancreatic acinar cell.