Previously it was known that cytosolic Ca(2+) elevation was involved in regulating UV- or TNFalpha-induced apoptosis. Here, we reported new evidence that mitochondrial Ca(2+) signal is also involved in the apoptotic process. First, using living cell imaging techniques, we observed multiple mitochondrial Ca(2+) spikes during the early stage of UV- or TNFalpha-induced apoptosis. Second, the mitochondrial Ca(2+) spikes were synchronous with cytosolic Ca(2+) spikes observed in apoptosis, which preceded cytochrome c (cyt-c) release. Third, blocking the mitochondrial Ca(2+) elevation by applying a mitochondrial uniporter inhibitor could suppress UV-induced apoptosis in HeLa cells. Finally, overexpressing an anti-apoptotic protein, Bcl-2, could suppress the mitochondrial Ca(2+) elevation. Furthermore, it appeared that the elevation of mitochondrial Ca(2+) during apoptosis was caused by a direct coupling between endoplasmic reticulum (ER) and mitochondria through IP(3) receptors. Taken together, these findings suggest that Ca(2+) mobilization from ER to mitochondria can play a significant role in the apoptotic signaling pathway.