India has one of the world's highest incidences of oral cancer. It is believed that the widespread habit of betel quid chewing is an important risk factor as it exposes the oral mucosa to known carcinogens. It also induces physical abrasions, which may create mitogenic environments during wound healing as gateways for infections. A recent study from our laboratories identified human papillomavirus (HPV) DNA, mostly of the high-risk types HPV-16 and HPV-18, in 67 of 91 oral cancer lesions from a cohort of Indian patients consisting mostly of betel quid users. This suggested a viral etiology of some lesions but tumorigenesis in the absence of viruses in other lesions. Here, we examined whether the p53 gene, whose function is abrogated by the product of the HPV gene E6, would be mutated in those oral cancers that were free of HPV DNA, and we found point mutations at known hot spots for mutational alteration of p53 in 4 of 23 lesions. We also considered the possibility that p21, a target of regulation by the p53 protein, may be mutationally altered in tumors with a functional p53 gene. While we did not identify mutations in the p21 gene, 6 of 11 lesions contained a polymorphism that may be associated with cancer. Interestingly, 3 of 23 lesions had mutations in the p16 gene, a third regulator of the cell cycle which is frequently mutated in melanoma but rarely in other cancers, with 1 lesion even having a mutation in the p53 as well as in the p16 gene. Our data point to p53 and p16 as gene targets of oral carcinogenesis, with chemicals in the betel quid possibly functioning in these tumors as carcinogens.