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The synergistic effect of ultrasound and certain chemicals on cells is known as sonodynamic therapy (SDT). It has been reported that the direct sonochemical and subsequent redox reactions induced by SDT treatment can lead to apoptotic cell death. However, the detailed biologic mechanism about it is not well understood until now. In this study the effect of low-intensity ultrasound on Hepatoma-22 cells (H22) in the presence of the sonosensitizing drug protoporphyrin IX (PpIX) was evaluated at different incubation times after sonication. Trypan blue exclusion was used to detect cell viability. The presence of apoptotic cells was identified by 4'-6-diamidino-2-phenylindole (DAPI) nuclear staining and transmission electric microscope (TEM) observation. An inverted confocal laser scanning microscope was used to detect the release of mitochondrial protein cytochrome c (Cyt c) and the redistribution of Bcl-2 family proteins Bid and Bax. Additionally, the generation of intracellular reactive oxygen species (ROS) and the loss of mitochondria membrane potential (MMP) were quantificationally measured by a fluorescence microplate reader. The results indicated that the synergistic cytotoxicity of PpIX and ultrasound increased in a time-dependent manner and the mitochondria damage may be the main factor for sonodynamically induced apoptosis by PpIX in H22 cells.