Objective To assess the effects of nuclear translocation of tissue transglutaminase (TTG) and the release of cytochrome C on hepatocyte apoptosis and to reveal the mechanism of signal transduction of early apoptosis in injured hepatocytes. Methods Hepatocytes isolated from tissue transglutaminase gene knock-out rats and mice were stimulated with ethanol. Proteins from whole cell, cytoplasm and nuclei were extracted for determination of TTG activity by 14 C-putrescine incorporation. Distribution of TTG throughout the entire cell, as well as just nucleus was observed under a confocal scanning microscope. The amount of cytochrome C released from mitochondria was determined by ELISA. Cell apoptosis was observed by fluorescent cytochemistry.Results TTG activity in whole cells and nuclei was significantly increased after the hepatocytes were treated with ethanol. Cytochrome C release was remarkably increased in the cells isolated from rat and wild-type mouse after treatment with ethanol but not in TTG gene knock-out mice. Cellular apoptosis appeared in hepatocytes isolated from rats and wild-type mice but not in the hepatocytes from TTG gene knock-out mice after stimulation with ethanol.Conclusions Increased TTG in hepatocytes can be translocated into the nucleus and promote release of mitochondrial cytochrome C into the cytoplasm. Passing through a series of signal pathways, hepatocyte apoptosis is induced eventually.