It has been previously shown that Walker 256 tumor cells express a high content of the anti-apoptotic protein Bcl-2 which protects mitochondria against the damaging effects of Ca(2+). In the present study, we analyze H(2)O(2)-induced apoptotic death in two different types of tumor cells: Walker 256 and SCC-25. Treatment with H(2)O(2) (4mM) increased reactive oxygen species generation and the concentration of cytosolic free Ca(2+). These alterations preceded apoptosis in both cell lines. In Walker cells, which show a high Bcl-2/Bax ratio, apoptosis was dependent on calcineurin activation and independent of changes in mitochondrial membrane potential (DeltaPsi(m)), as well as cytochrome c release. In contrast, in SCC-25 cells, which show a lower Bcl-2/Bax ratio, apoptosis was preceded by a decrease in DeltaPsi(m), mitochondrial permeability transition, and cytochrome c release. Caspase-3 activation occurred in both cell lines. The data suggest that although the high Bcl-2/Bax ratio protected the mitochondria of Walker cells from oxidative stress, it was not sufficient to prevent apoptosis through calcineurin pathways.39186-94