Oxidative stress plays an important role in the pathogenesis of acute pancreatitis. Increased generation of reactive oxygen species is a well known initiator of apoptosis and has been shown to induce apoptosis in mouse pancreatic acinar cells [1]. Opening of the permeability transition pore, release of cytochrome c, and activation of caspase-9 occurs in these cells in response to oxidative stress; however, a small population of cells can still undergo apoptosis when the opening of the permeability transition pore is blocked. We investigated the possible role and mechanism of action of the alternative apoptotic pathways versus the intrinsic pathway in pancreatic acinar cells exposed to oxidant stress. Mouse pancreatic acinar cells (freshly isolated from CD1 mice) were loaded with a fluorescent substrate for caspase-9 or caspase-8. Using confocal microscopy, fluorescence of cleaved substrate was imaged in real time in response to the oxidant menadione (30 µM). Cells were positive for caspase activation when fluorescence was higher than the average fluorescence of control cells plus two standard deviations. Caspase-9 or caspase-8 activity was examined in control cells and cells pre-treated with 25 μM BAPTA-AM to prevent cytoplasmic calcium elevations, 200 µM TPEN to reduce calcium within the endoplasmic reticulum, 50 µM bongkrekic acid to block opening of the mitochondrial permeability transition pore, 10-40 µM caspase-9 inhibitor, or 50 µM GPN to disrupt lysosomes. An unpaired, two-tailed Student’s t test was used for statistical comparisons between treatment groups (p<0.05 was significant). Caspase-9 was activated within a few minutes (t½ = 129 ± 43 s; n=12) after administration of menadione. Activation of caspase-9 was significantly inhibited in cells pre-treated with BAPTA-AM, but no change was observed in the presence of 200 μM of TPEN (decreased ER calcium content as measured with Mag-Fluo4). Caspase-8 was activated (t½) within 26 ± 3 min (n=8) after treatment with menadione in 21 ± 3% of cells (n=716). Activation of caspase-8 was not altered by inhibition of the intrinsic apoptotic pathway with bongkrekic acid or caspase-9 inhibitor. Caspase-8 activation was also not changed in the presence of BAPTA-AM. However, caspase-8 activation was significantly reduced when lysosomes of the cells were destroyed with GPN. Both, the intrinsic and in some cells extrinsic apoptotic pathways are rapidly activated in response to oxidative stress in the pancreatic acinar cell. Caspase-9 activation is calcium-dependent, however, does not require for activation full ER calcium stores. Caspase-8 is activated independently of the intrinsic apoptotic pathway and does not require the increase of cytoplasmic calcium. In contrast, caspase-8 requires functional lysosomes for oxidative stress-induced activation in pancreatic acinar cells.