Pioglitazone (PIO), a member of the thiazolidinedione class of antidiabetic agents, specifically targets insulin resistance. Drugs of this class act as ligands for the gamma subtype of the peroxisome proliferator-activated receptor. Although troglitazone, another drug in this class, displayed unacceptable hepatotoxicity, PIO was approved for human use by the U.S. Food and Drug Administration. To our knowledge, there are no published reports on the genotoxicity of PIO; however, the package insert indicates that it has minimal genotoxicity. In this study, we used the comet assay to investigate the DNA damage in the peripheral blood and liver cells of rats treated with PIO. Sixteen male Sprague-Dawley rats were randomly distributed into four groups, and dosed daily for 14 days by oral gavage with 0, 10, 20, and 40 mg/kg/day PIO. A dose-dependent increase in DNA damage, as assessed by % tail DNA, was observed in both hepatocytes and blood lymphocytes of the PIO-treated groups, with significant increases detected between the rats treated with all the doses of PIO and the control, and between the rats treated with different PIO doses (P < 0.005 to P < 0.0001). Treating nuclei from the exposed animals with an enzyme cocktail containing Fpg and Endonuclease III prior to performing the comet assay increased the level of DNA damage, which reflects oxidized purine and pyrimidine. Taken together, our data indicate that PIO is able to dose-dependently induce DNA damage in both the liver and blood lymphocytes of rats, which is partially due to the generation of oxidative lesions.