Background: Cell cycle checkpoints function to maintain genetic stability by providing additional time for repair of DNA damage and completion of events that are necessary for accurate cell division. Some checkpoints, such as the DNA damage G(1) checkpoint, are dependent on p53, whereas other checkpoints, such as the decatenation G(2) checkpoint, are not. Because bladder transitional cell carcinomas (TCCs) often contain numerous chromosomal aberrations and appear to have highly unstable genomes, we analyzed cell cycle checkpoint functions in a panel of TCC lines. Methods: Cell cycle arrest was induced in normal human fibroblasts (NHF1-hTERT) and normal human uroepithelial cells (HUCs), and TCC lines and checkpoint functions were quantified using flow cytometry and fluorescence microscopy. The inducers and checkpoints were ionizing radiation (i.e., DNA damage) (G(1) and G(2) checkpoints), the mitotic inhibitor colcemid (polyploidy checkpoint), or the topoisomerase 11 catalytic inhibitor ICRF-193 (decatenation G(2) checkpoint). Four of the five TCC lines expressed mutant p53. Results: HUCs had an effective G(1) checkpoint response to ionizing radiation, with 68% of cells inhibited from moving from G(1) into S phase. By contrast, G(1) checkpoint function was severely attenuated (<15% inhibition) in three of the five TCC lines and moderately attenuated (<50% inhibition) in the other two lines. NHF1-hTERT had an effective polyploidy checkpoint response, but three of five TCC lines were defective in this checkpoint. HUCs had effective ionizing radiation and decatenation G(2) checkpoint responses. All TCC lines had a relatively effective G(1) checkpoint response to DNA damage, although the responses of two of the TCC lines were moderately attenuated relative to HUCs. All TCC lines had a severe defect in the decatenation G(2) checkpoint response. Conclusion: Bladder TCC lines have defective cell cycle checkpoint functions, suggesting that the p53-independent decatenation G(2) checkpoint may cooperate with the p53-dependent G(1) checkpoints to preserve chromosomal stability and suppress bladder carcinogenesis.