Chemicals that induce tumors in the urinary bladder in long-term toxicity tests often induce hyperplasia, a pre-cancerous lesion in subacute toxicity tests. Determining whether or not the mechanism by which hyperplasia induced in the urinary bladder is due to genotoxicity at an early stage is important for the extrapolation of carcinogenicity to humans. An in vivo comet assay is suitable for shortterm evaluation of urinary bladder genotoxicity. 2-Acetylaminofluorene (2-AAF), diuron, and terephthalic acid (TPA) are known to induce tumors in rat bladder by different mechanisms in long-term toxicity tests, but so far there are no reports on the bladder comet assay for the evaluation of these compounds in short-term toxicity tests. Here, a comet assay in rat urinary bladder for short-term evaluation was assessed by using rat strains in which hyperplasia induction has been reported for diuron and TPA, both of which induce hyperplasia in subacute toxicity tests, and rat strains in which tumors have been observed in longterm toxicity tests of 2-AAF. Regarding the respective DNA damaging properties of these compounds, the result was positive for 2-AAF, a typical genotoxic urinary bladder carcinogen, but negative for diuron and TPA, which induce hyperplasia through the chemical action of metabolites and through physical cytotoxicity due to urinary crystals, respectively. Thus, the comet assay of rat urinary bladder as a short term in vivo genotoxicity test is considered to be a useful and convenient method for the short-term evaluation of the genotoxicity of compounds which induce hyperplasia in the urinary bladder.