Short-term exposure (43 C for 15 min) of the rat testis to mild heat results, within 6 h, in stage- and cell-specific activation of germ cell apoptosis. Initiation of apoptosis was preceded by a redistribution of Bax from a cytoplasmic to paranuclear localization in heat-susceptible germ cells. Here we show that the relocation of Bax is accompanied by cytosolic translocation of cytochrome c and is associated with activation of the initiator caspase 9 and the executioner caspases 3, 6 and 7, and cleavage of PARP. Furthermore, early in apoptosis, a significant amount of Bax also accumulates in endoplasmic reticulum (ER) as assessed by Western blot analyses of fractionated testicular lysates. In additional studies, using the FasL-defective gld mice, we have shown that heat-induced germ cell apoptosis is not blocked, thus providing evidence that the Fas signaling system may be dispensable for heat-induced germ cell apoptosis in the testis. Taken together, these results demonstrate that the mitochondria- and possibly also ER-dependent pathways are the key apoptotic pathways for heat induced germ cell death in the testis.