The frequent loss or promoter methylation of the tumor suppressor gene p16 in head and neck cancer suggests an etiologic role of p16 in this disease. Two adjacent polymorphisms of p16 exon 3, C540G and C580T, were identified recently. C540G is associated with low expression of p53, and both polymorphisms are associated with tumor aggressiveness, suggesting a possible functional relevance. We hypothesized that these two polymorphisms, particularly their haplotypes, are associated with the risk of developing squamous cell carcinoma of the head and neck (SCCHN). To test this hypothesis, we conducted a hospital-based case-control study of 208 patients with SCCHN and 224 cancer-free control subjects to evaluate the association between p16 genotypes/haplotypes and the risk of SCCHN, using a PCR-single strand conformation polymorphism-based genotyping assay. However, our results suggested that no significant differences exist in the distribution of p16 C540G and C580T genotypes between cases and controls. For the C540G polymorphism, the CC, CG, and GG genotype frequencies were 76.9%, 22.1%, and 1.0%, respectively, in the cases, compared with 76.8%, 21.9%, and 1.3%, respectively, for the controls. For the C580T polymorphism, the CC, CT, and TT genotype frequencies were 83.6%, 15.9%, and 0.5%, respectively, in the cases, compared with 82.6%, 16.5%, and 0.9%, respectively, for the controls. The frequencies of three predominant 540C/580C, 540G/580C, and 540C/580T haplotype alleles were distributed similarly in the cases (79.6%, 12.0%, and 8.4%) and in the controls (78.6%, 12.3%, and 9.1%). None of these differences were statistically significant. We conclude that these polymorphic p16 genotypes or haplotypes may not play a major role in the etiology of SCCHN, if any. However, our limited sample size and power call for larger studies for additional verification of our findings.