The pancreatic acinar cells are an excellent model to study intracellular Ca²⁺ signalling. We have shown previously that in normal pancreatic acinar cells (freshly isolated from humanely killed CD1 mice) the oxidant menadione evokes repetitive cytosolic Ca²⁺ spikes, partial mitochondrial depolarisation, cytochrome c release and apoptosis. The physiological agonists acetylcholine (ACh) and cholecystokinin also evoke cytosolic Ca²⁺ spikes, but neither depolarise mitochondria nor induce apoptosis. Ca²⁺ spikes induced by low ACh concentrations are confined to the apical secretory pole of the cell by the buffering action of peri-granular mitochondria. Menadione prevents mitochondrial Ca²⁺ uptake, permitting rapid spread of Ca²⁺ throughout the cell and activation of caspase-9 and -3 (Gerasimenko et al. 2002). The Ca²⁺ chelator BAPTA prevents cytosolic Ca²⁺ spiking, blocks the menadione-elicited mitochondrial depolarisation and blocks menadione-induced apoptosis. Menadione induced apoptosis in this cell type is calcium dependent and rapid, with cytochrome c release occurring at 2 min. Our recent studies show real time activation and spatial distribution of caspase-9 and -3 using confocal microscopy of fluorescent caspase substrates. Here, we show conclusively that caspase-9 activation in response to menadione is rapid (the time to 1/2 max. activation (t_1/2) was 129 ± 43 s; n=12) and calcium dependent and that caspase-9 may be activated at or close to mitochondria in response to menadione in pancreatic acinar cells.