The transcriptional silencing of some cell cycle inhibitors and tumor suppressors, such as p16 and retinoic acid receptor beta(2), by DNA hypermethylation at CpG islands is commonly found in human oral squamous carcinoma cells. We examined the effects of the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza; 0.25 mg/kg body weight), all-trans retinoic acid (RA; given at 100 microg/kg body weight and 1 mg/kg body weight), and the combination of 5-Aza and the low-dose RA on murine oral cavity carcinogenesis induced by the carcinogen 4-nitroquinoline 1-oxide (4-NQO) in a mouse model. All the drug treatments were done for 15 weeks after a 10-week 4-NQO treatment. Mice in all drug treatment groups showed decreases in the average numbers of neoplastic tongue lesions. The combination of 5-Aza and RA effectively attenuated tongue lesion severity. Although all drug treatments limited the increase in the percentage of proliferating cell nuclear antigen-positive cells and the decrease in the percentage of p16-positive cells caused by the 4-NQO treatment in mouse tongue epithelial regions without visible lesions and in the neoplastic tongue lesions, the combination of 5-Aza and RA was the most effective. Collectively, our results show that the combination of a DNA demethylating drug and RA has potential as a strategy to reduce oral cavity cancer in this 4-NQO model.