The cyclin-dependent kinase inhibitor gene p21(Waf1/Cip1) plays a central role in inducing cellular growth arrest, terminal differentiation, and apoptosis. Alterations in this gene may adversely affect regulation of these processes and increase susceptibility for cancer. We have recently reported a novel polymorphism in the p21(Waf1/Cip1) gene in the Indian population and its association with esophageal cancer. An A-->G transition at codon 149 resulted in amino acid substitution from aspartate to glycine in the proliferating cell nuclear antigen binding COOH-terminal domain of p21(Waf1/Cip1) that may affect PCNA-p21(Waf1/Cip1) interactions, thereby affecting regulation of cellular proliferation, and may increase susceptibility for development of cancer. In a parallel study in our laboratory, we searched for putative p21(Waf1/Cip1) mutations in oral premalignant and malignant lesions. No somatic mutation was detected in exon 2 of p21(Waf1/Cip1). Interestingly, a codon 149 polymorphism variant (A-->G) was identified in 11 of 30 (37%) premalignant lesions (7 of 19 hyperplastic lesions and 4 of 11 dysplastic lesions) and 11 of 30 (37%) squamous cell carcinomas (SCCs). This codon 149 variant was also identified in paired lymphocytes of all of the patients with premalignant lesions and SCCs harboring the variant allele, suggesting the occurrence of a polymorphism. Lymphocyte DNA isolated from 50 unrelated age- and gender-matched healthy subjects was screened for this polymorphism. Seven of 50 (14%) normal controls harbored the A-->G codon 149 variant allele. Immunohistochemical analysis of p21(Waf1/Cip1) protein expression showed immunoreactivity in 19 of these 30 (63%) oral premalignant lesions and 16 of 30 (53%) SCCs. The most intriguing features of the study were: (a) the significant increase in frequency of this polymorphism not only in patients with oral SCCs (P = 0.038), but also in patients with premalignant lesions (P = 0.038), compared with normal controls; and (b) the significantly higher frequency of p21(Waf1/Cip1) variants (codon 149) in oral premalignant lesions (10 of 11 cases) and SCCs (11 of 11 cases) with wild-type p53 (P = 0.045) than in lesions with p53 mutations, suggesting that this polymorphism affects the p53 pathway and may play a vital role in oral tumorigenesis. Furthermore, overexpression of p21 protein in oral lesions harboring missense mutations in the p53 gene suggest a p53-independent role for p21 in the pathogenesis of oral cancer.