Array-based comparative genomic hybridization (array-CGH) has good potential for the high-throughput identification of genetic aberrations in cell genomes. In the course of a program to screen a panel of 21 oral squamous-cell carcinoma (OSCC) cell lines for genome-wide copy-number aberrations by array-CGH using our in-house bacterial artificial chromosome arrays, we identified a frequent homozygous deletion at 4q35 loci with approximately 1 Mb in extent. Among the seven genes located within this region, the expression of the melatonin receptor 1 A (MTNR1A) messenger RNA (mRNA) was not detected or decreased in 35 out of the 39 (89%) OSCC cell lines, but was detected in immortalized normal oral epithelial cell line, and was restored in gene-silenced OSCC cells without its homozygous loss after treatment with 5-aza-2'-deoxycytidine. The hypermethylation of the CpG (cytosine and guanine separated by phosphate) island in the promoter region of MTNR1A was inversely correlated with its expression in OSCC lines without a homozygous deletion. Methylation of this CpG island was also observed in primary OSCC tissues. In an immunohistochemical analysis of 50 primary OSCC tumors, the absence of immunoreactive MTNR1A was significantly associated with tumor size and a shorter overall survival in patients with OSCC tumors, and seems to be an independent prognosticator in a multivariate analysis. Exogenous restoration of MTNR1A expression inhibited the growth of OSCC cells lacking its expression. Together with the known tumor-suppressive function of melatonin and MTNR1A in various tumors, our results indicate MTNR1A to be the most likely target for epigenetic silencing at 4q35 and to play a pivotal role during oral carcinogenesis.