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Lethal toxin (LT) from Clostridium sordellii (strain IP82) inactivates in glucosylating the small GTPases Ras, Rap, Ral and Rac. In the present study we show that LT-IP82 induces cell death via an intrinsic apoptotic pathway in the myeloid cell-line HL-60. LT-IP82 was found to disrupt mitochondrial homeostasis as characterized by a decrease in mitochondrial transmembrane potential and cardiolipin alterations, associated with the release of cytochrome c in the cytosol. Time-course studies of caspase activation revealed that caspase-9 and caspase-3 were activated before caspase-8. Moreover, although LT-IP82-induced cell death was abrogated by caspase-inhibitors, these inhibitors did not suppress mitochondrial alterations, indicating that caspase activation occurs downstream of mitochondria. Protection of mitochondria by Bcl-2 overexpression prevented mitochondrial changes as well as apoptosis induction. Furthermore, evidence is provided that LT-IP82-induced apoptosis is not a consequence of cortical actin disorganization, suggesting that Rac inactivation does not initiate the apoptotic process. Cell exposure to LT-IP82 leads to a co-localization of the toxin with a mitochondrial marker within 2 h. Therefore, we suggest that LT-IP82 could act at the mitochondrion level independently of its enzymatic effect on small GTPases.