Although reactive oxygen species and calcium have been separately reported to be important mediators of apoptosis, little correlation between these two mediators has been reported. Here, we focused onto the role of calcium on H₂O₂-induced mitochondrial apoptosis in AR42J cells. AR42J cells were cultured in RPMI supplemented with glutamine, fetal bovine serum and antibiotics at 37ordm;C in humidified incubator (5% CO₂). Caspase-3 activity was determined from the cleavage of specific fluorogenic substrate (AC-DEVD-AMC), and cytochrome c release was detected by Western blotting in samples from the mitochondrial and cytosolic fractions. Treatment of AR42J cells with H₂O₂ for 24 hours induced a concentration-dependent activation of caspase-3. Cell stimulation with H₂O₂ caused a detectable activation of caspase-3 at 1 mM with a 147.6 ± 46 %, and the maximum effect was obtained at a dose of 100 mM (211.2 ± 82 %). The effect of H₂O₂ on caspase-3 activation was also time-dependent, reaching a maximal caspase activity after 24 h of stimulation (198.4 ± 47 %) at the concentration of 10 mM. Cell exposure to 10 mM H₂O₂ or 1 mM tapsigargin for 24 h results in cytochrome c release, however dimethyl BAPTA loading did not modify significantly H₂O₂- and thapsigargin-induced caspase-3 activation and cytochrome c release. Furthermore, pretreatment with Ru360, a blocker of calcium uptake into mitochondria, was able to reduce H₂O₂- and thapsigargin-evoked caspase-3 activation. We conclude that H₂O₂-evoked mitochondrial apoptosis in AR42J cells is dependent of calcium uptake into mitochondria. This work was supported by MEC-DGI grant BFU2004-0165.