Tributyltin(TBT) used world-wide in antifouling paints for ships is a widespread environmental pollutant and cause reproductive organs atrophy in rodents. At low doses, antiproliferative modes of action have been shown to be involved, whereas at higher doses apoptosis seems to be the mechanism of toxicity in reprodctive organs by TBT. In this study, we investigated that the mechnisms underlying DNA fragmentation induced by TBT in the rat leydig cell line, R2C. Effects of TBT on intracellular Ca^(2+) level and reactive oxygen species(ROS) were investigated in R2C cells by fluorescence detector. TBT significantly induced intracellular Ca^(2+) level in a time-dependent manner. The rise in intracellular Ca^(2+) level was followed by a time-dependent generation of reactive oxygen species(ROS) at the cytosol level. Simultaneously, TBT induced the release of cytochrome c from the mitochondrial membrane into the cytosol. Furthermore, ROS production and the release of cytochrome c were reduced by BAPTA, an intracellular Ca^(2+) chelator, indicating the important role of C^(2+) in R2C during these early intracellular events. In addition, Z-DEVD FMK, a caspase-3 inhibitor, decreased apoptosis by TBT. Taken together, the present results indicated that the apoptotic pathway by TBT might start with an increase in intracellular Ca^(2+) level, continues with release of ROS and cytochrome c from mitochondria, activation of caspases, and finally results in DNA fragmentation.