We showed previously that a cytosolic Ca2+ signal is involved in regulating UV-induced apoptosis in HeLa cells. In this study, we found evidence that this Ca2+ signal occurs upstream of the release of cytochrome c from mitochondria. First, when we abolished [Ca2+](i) increases by injecting BAPTA or heparin into UV-treated HeLa cells, cytochrome e release was either blocked or severely delayed. Second, using a living cell imaging technique, we observed a series of transient [Ca2+](i) increases (typically lasting about 4060 s) in many apoptotic cells induced by either UV- or TNFalpha-treatment. Third, using GFP-tagged cytochrome c, we found that the Ca2+ spikes appear in a time window before cytochrome c was released. Finally, by fixing the TNFalpha-treated cell at the time when it started to display Ca2+ spikes, we examined the distribution of its endogenous cytochrome c using immunostaining. We found that cytochrome c was not yet released from mitochondria. These findings suggest the existence of certain apoptotic pathways, in which an early Ca2+ signal is activated upstream of cytochrome c release. (C) 2002 Elsevier Science (USA). All rights reserved.