Matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) play an important role in several stages of cancer initiation and development. Single nucleotide polymorphisms identified in the promoters of MMP2 (-1306C-->T) and TIMP2 (-418G-->C) abolish the Sp1-binding site and thus may down-regulate expression of the genes. We examined the contribution of these polymorphisms to susceptibility and aggressiveness of head and neck squamous cell carcinoma (HNSCC). MMP2 genotypes were determined by PCR-based allele-specific refractory mutation analysis and TIMP2 genotypes identified by PCR-RFLP in a panel of HNSCC cell lines and in 239 head and neck cancer patients and 250 frequency matched controls in an ethnic Thai population. We found that subjects with the MMP2 CC genotype was associated with significantly increased risk [adjusted odds ratio (OR), 1.97; 95% confidence interval (95% CI), 1.23-3.15] for developing HNSCC compared with those with the variant genotype (-1306CT or TT). For TIMP2, a moderately increased risk of the cancer (OR, 1.43; 95% CI, 0.98-2.08) was also associated with the variant allele (-418GC or CC), compared with the GG common allele. Furthermore, the polymorphisms in both genes showed some additive effect and the highest risk for head and neck cancer was observed in those with MMP2 CC genotype and TIMP2 variant GC or CC genotype (OR, 2.34; 95% CI, 1.31-4.18). A correlation between promoter polymorphisms and the levels of mRNA expression in cell lines and cancer tissues was found. Finally, the MMP2 CC genotype was correlated with adverse clinicopathological variables. These findings suggest that the genetic polymorphisms in the promoters of MMP2 and TIMP2 may be associated with the development and aggressiveness of HNSCC.