Telomerase, a reverse transcriptase that maintains telomere length, is highly expressed in tumor cells. Consequences of telomere dysfunction may include genomic instability, senescence or p53-induced apoptosis. Moreover, oxidative stress is known to play a critical role in DNA damage and apoptotic events. In order to investigate the relationship between oxidative stress in tumor cell death and telomerase transcription, Walker 256 tumor cells were treated with 4 mM H₂O₂. After 3 hours, almost 50% of the cells were Annexin-V positive as observed by flow cytometry. We detected significant increase in reactive oxygen species (ROS) production associated with increase in [Ca²⁺]_cytosol in treated cells. Either BAPTA, Cyclosporin A or FK-506 prevented decrease in cell viability, while Bongkrekic acid did not. This suggests that both Ca²⁺ and calcineurin but not mitochondrial permeability transition pore are involved in this apoptotic process. In accordance, no mitochondrial cytochrome c release was observed by Western blotting. Caspase-3 activity was significantly enhanced in treated cells despite the absence of cytochrome c release. H₂O₂ treatment induced significant decrease of telomerase catalytic subunit (TERT) mRNA transcription as observed by semi-quantitative RT-PCR. The addition of pifithrin-alpha (PFT), a synthetic p53 inhibitor, significantly decreased apoptosis in treated cells, evidencing the participation of p53 tumor suppression protein in this cell death process. Together, these results suggest that oxidative stress leads Walker 256 tumor cells to p53-dependent apoptosis, probably via disturbances in both [Ca²⁺]_cytosol homeostasis and in telomerase transcription. Moreover, calcineurin and caspase-3 activation seems to participate in H₂O₂-induced apoptotic pathway in these cells.