Thimerosal is one of the most widely used preservatives and is found in a variety of biological products, including vaccines, contact lens cleaning solutions, and cosmetics. It has been reported to have harmful effects on epithelial tissues, such as causing conjunctivitis or contact dermatitis. However, the molecular mechanism of its toxicity has not been characterized using epithelial tissues. In the present study, we report that reactive oxygen species play a key role in thimerosal-induced cytotoxicity in HeLa S epithelial cells. Thimerosal significantly reduced HeLa S cell viability and it was associated with a decrease in intracellular glutathione levels. Flow cytometric cell cycle analysis showed a marked increase in the hypodiploidic cell population, indicating apoptosis of thimerosal-treated cells. The apoptotic cell death of epithelial cells was confirmed by observing a significant increase of caspase-3 activity in the cytosolic fraction of the treated cells. Thimerosal also induced a concentration-dependent increase of genomic DNA fragmentation, a biochemical hallmark of apoptosis. Hoechst 33342 nuclear staining demonstrated apoptotic-fragmented multinuclei in thimerosal-treated cells. All the thimerosal-mediated toxic responses observed in the present study were almost completely suppressed by pretreating cells with N-acetyl-l-cysteine, a radical scavenger. Taken together, these results suggest for the first time that epithelial cytotoxicity of thimerosal is mediated by oxidative stress.