Tributyltin (TBT) used world-wide in antifouling paints for ships is a wide-spread environmental pollutant. At low doses, antiproliferative modes of action have been shown to he involved, whereas at higher doses apoptosis seems to he the mechanism of toxicity in reproductive organs by TBT. In this study, we investigated that the mechanisms underlying apoptosis induced by TBT in R2C cell. Effects of TBT on intra-cellular Ca^(2+) level and reactive oxygen species (ROW 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 (ROW 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 CaZ+ chelator, indicating the important role of Ca`+ in R2C during these early intracellnlar events. In addition, Z-DEW 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 Caz+ level, continues with release of ROS and cytochrome c from mitochondria, activation of caspases, and finally results in DNA fragmentation.