The medium-term tongue carcinogenesis assay is a useful model for studying oral squamous cell carcinomas phase by phase. The aim of the present study was to investigate the expression of p53 by immunohistochemistry and examine the DNA sequence of exons 5, 6, 7, and 8 of Tp53 for mutations during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide (4NQO). A total of 30 male Wistar rats were treated with 4-nitroquinoline 1-oxide in their drinking water for 4, 12, and 20 weeks at a dose of 50 ppm. Ten animals were used as negative controls. No histopathological changes in the tongue epithelia were observed in the control group or in the treatment group after 4 weeks of 4NQO. Following 12 weeks of treatment, hyperplasia as well as epithelial dysplasia was found in both mild and moderate forms. At 20 weeks, moderate and/or severe oral dysplasia and squamous cell carcinoma of the tongue were found, and the majority of animals had squamous cell carcinoma. The levels of p53 protein were increased (p < 0.05) in pre-neoplastic lesions and in squamous cell carcinomas in some of the tumor cells in squamous cell carcinomas. No mutations were found in any of the exons that were evaluated after the 4-, 12-, or 20-week treatments. Taken together, our results suggest that p53 expression may be an important event in the malignant conversion, whereas Tp53 mutations are not involved in the multi-step tongue carcinogenesis of Wistar rats induced by 4NQO.