Abstract Introduction: Panels of hypermethylated genes have been proposed as diagnostic markers in primary head and neck squamous cell carcinoma using a pharmacological unmasking expression microarray approach. Such experiments are performed in cancer cell lines, mostly with poor relevance when extrapolating to primary cancers. Objective: To overcome this lack of relevance we used two unbiased genome-wide DNA methylation platforms that represent 28K CpG Islands and the promoter regions of 14K-17K genes to identify hypermethylated genes in Oral Squamous Cell Carcinoma (OSCC), which also exhibited downregulated expression in the Gene Expression Omnibus (GEO) repository. Methods: DNA isolated from normal, premalignant lesions and squamous cell carcinoma lesions of the oral cavity was: a) bisulfite treated and hybridized to Infinium arrays; b) enriched with Methylated DNA Immunoprecipitation (MeDIP) and hybridized to Nimblegen 385K tiling arrays. Bioinformatics strategies were used for background correction, array normalization and data analysis of differentially methylated genomic regions between tumor and normal tissue. Genes identified as hypermethylated in cancer with methylation platforms were cross-validated against expression profiles published in the GEO public data repository. Quantitative Methylation Specific PCR (QMSP) was then used to validate candidate genes that were both, hypermethylated and downregulated in cancer. Results: The two unbiased microarray platforms were successful in identifying three novel genes hypermethylated and down regulated in OSCC: MCAM, EDNRB and CALCA. EDNRB, MCAM and CALCA expression was downregulated in HNSCC when compared to normal tissue. A twofold or higher downregulated expression was seen for EDNRB (59%) than for MCAM (55%) and CALCA (55%). The sensitivity of each gene was higher for EDNRB (82%) than for MCAM (60%) and CALCA (76%). The specificity was also higher for EDNRB (86%) than for CALCA (71%), and MCAM (57%). KIF1a had both high sensitivity (82%) and specificity (86%) but there was no expression data in HNSCC available for this gene in the GEO repository. The Receiver Characteristic Operator Curves (ROC) for individual genes revealed area under the curve values (AUC) for: EDNRB (0.88), CALCA (0.85), KIF1a (0.84), and MCAM (0.61). Methylation of three genes was found in 71% of tumor and 0% of normal tissue. Methylation of two genes was found in 82% of tumor and 29% of normal tissue. Conclusion: We successfully demonstrated that unbiased genome-wide methylation arrays paired with publicly available expression data identify panels of relevant hypermethylated genes in primary OSCC. MCAM, KIF1a, EDNRB and CALCA were identified as hypermethylated in OSSC tumor tissue, when compared to normal tissue. These results should be validated in a Phase II Biomarker Development Study. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2924.