We previously reported that immunostaining for γ-H2AX, a biomarker of DNA damage, in the rat urinary bladder is useful for early detection of bladder carcinogens in 28-day toxicity studies. Here, we aimed to examine the dose dependency of γ-H2AX formation in the urinary bladder of rats. Male F344 rats (aged 6 weeks) were orally administered N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN; 0%, 0.0001%, 0.001%, 0.01%, 0.02%, or 0.05% in drinking water), a genotoxic bladder carcinogen, and melamine (0%, 0.3%, 1.0%, or 3.0% in the diet), a nongenotoxic bladder carcinogen, for 2 days or 4 weeks. Immunohistochemical analysis showed that γ-H2AX- and Ki67-positive epithelial cells in the bladder urothelium were significantly increased, with a clear dose dependency, in both BBN- and melamine-treated groups. Additionally, γ-H2AX formation was detected from the lower-dose group, without increased Ki67 expression or histopathologic findings. The ratios of γ-H2AX-positive cells at week 4 in both BBN- and melamine-treated groups were higher than those on day 2, indicating the time-dependent increase in γ-H2AX formation. Immunofluorescence double-staining revealed that γ-H2AX single-positive cells without Ki67 expression were often found in the urothelium of BBN-treated rats, whereas most γ-H2AX-positive cells were Ki67-positive in the melamine group. Our results demonstrated that γ-H2AX formation in the urinary bladder increased in a clear dose-dependent manner and that γ-H2AX immunostaining has the potential to detect bladder carcinogens after a 2-day administration. Furthermore, the association of genotoxic mechanisms in bladder carcinogenesis could be determined by analyzing the colocalization of γ-H2AX and Ki67 in the urothelium.