In this study, we first categorized nine squamous cell carcinoma (SCC) cell lines into two groups in terms of the expression of HLA-DR, -DP, and -DQ molecules. Subsequently, the expression of class II transactivator (CIITA) was studied in these cell lines, because it is widely accepted that the expression of MHC class II molecules is regulated by different types of CIITA transcripts that are initiated by distinct promoters. The majority of the SCC cell lines (six of nine) expressed HLA-DR molecules and CIITA promoter IV (pIV) transcripts in the presence of IFN-gamma. In contrast, three of the nine SCC cell lines were completely negative for class II molecules and all types of CIITA, suggesting epigenetic changes in the promoter region in these cells. Previously, methylation of CIITA pIV was reported to silence CIITA gene expression. We extensively studied the methylation status of CIITA pIV using a panel of 22 SCC cell lines. Remarkably, none of the SCC cell lines demonstrated hypermethylation at the site. In contrast, treatment with a histone deacetylation inhibitor in combination with IFN-gamma clearly restored the expression of the CIITA type IV gene in the HLA-DR-negative SCC cell lines, and the acetylation status of histone H3 examined by chromatin immunoprecipitation analysis was closely associated with the gene expression. Moreover, stable transfection of the CIITA gene into an HLA-DR-negative cell line restored constitutive expression of MHC class II molecules. Therefore, histone deacetylation, but not hypermethylation, modifies CIITA DNA and class II gene expression in SCC.