Cell death programmes executed in the pancreas under pathological conditions remain largely undetermined. We have defined mechanisms by which apoptosis is induced in pancreatic acinar cells by the oxidant stressor menadione. Real-time monitoring of initiator caspase activity showed that caspase-9 (66% of cells) and caspase-8 (15% of cells) were activated within 30 min of menadione administration, but no activation of caspase-2, -10 or -12 was detected. Interestingly, when caspase-9 activation was inhibited, activation of caspase-8 was increased. Half maximum activation of caspase-9 occurred within ~2 min and was identified at or in close proximity to mitochondria, whereas t_0.5 for caspase-8 occurred within ~26 min of menadione application, and was distributed homogeneously throughout cells. Caspase-9 but not caspase-8 activation was blocked completely by the calcium chelator BAPTA or bongkrekic acid, an inhibitor of the mitochondrial permeability transition pore. In contrast, caspase-8 but not caspase-9 activation was blocked by the destruction of lysosomes (pre-incubation with GPN, a cathepsin C substrate), loss of lysosomal acidity (bafilomycin A1), or inhibition of cathepsins -L or -D. Using pepstatin A-BODIPY FL conjugate we confirmed translocation of cathepsin D out of lysosomes in response to menadione. We conclude that the oxidative stressor menadione induces two independent apoptotic pathways within pancreatic acinar cells: the classical mitochondrial calcium-dependent pathway which is initiated rapidly in the majority of cells, and a slower, caspase-8-mediated pathway that depends on the lysosomal activities of cathepsins and is used when the caspase-9 pathway is disabled.