The aims of this study were to evaluate the DNA content of chemically-induced rat urothelial lesions and their relationship to the proliferation index and histological patterns. Sixty female Fisher 344 rats were divided randomly into six groups, four groups were exposed to N-butyl-N-(4-hydroxybutyl) nitrosamine for a period of 10 and 20 weeks, and two groups of ten rats were used as control animals. Paraffin sections were Feulgen stained and analyzed using DNA image cytometry analysis; histograms were classified as either diploid or aneuploid. Ki-67 immunoreactivity was determined by means of the streptavidin-biotin-complex immunoperoxidase method. All normal urothelium from the control groups were found to have diploid DNA content. The same histogram pattern was found in the simple hyperplasia group. As regards the other histological lesions, the frequency of the aneuploidy varied depending on the lesion type: 20% of aneuploidy were nodular hyperplasia, 32% of aneuploidy were dysplasias, 25% of aneuploidy were papilloma, 44% of aneuploidy were papillary neoplasm of low malignant potential, 22% of aneuploidy were low-grade papillary carcinoma, 100% of aneuploidy were high-grade papillary carcinoma and 100% of the aneuploidy were invasive carcinoma. Our results revealed the existence of a statistically significant relationship between DNA ploidy and histological pattern lesions (r=0.3, p<0.023). The Ki-67 proliferation index was significantly higher in aneuploid lesions than in diploid (r=0.56, p=0.01). There was also a statistically significant difference in the Ki-67 proliferation index in relation to the histopathological pattern (r=0.751, p<0.01). DNA content was associated with the Ki-67 proliferation index and histopathological grade. DNA content and prolife-ration index have critical roles to play during urothelial carcinogenesis.