The aim of this study was to investigate oxidative DNA damage during 4-nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinogenesis. For this purpose, male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution through their drinking water for 4, 12, and 20 weeks. Ten animals were used as negative control. The alkaline Comet assay modified with lesion-specific enzymes was used to detect single and double strand breaks, labile sites (SBs), and oxidised purines and pyrimidines. Although no histopathological abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure, oxidative DNA damage was detected in the ‘normal’ oral epithelium. In pre-neoplastic lesions and squamous cell carcinomas induced after 12 and 20 weeks following carcinogen exposure, respectively, oxidative DNA damage was also increased (P < 0.05) when compared to negative control. In conclusion, our results suggest that oxidative DNA damage is an early event during multistep carcinogenesis assay induced by 4NQO. This kind of approach should be considered to persons with high risk of oral cancer, such as in smokers or alcohol consumers.