Retinoic acid receptor-beta(2) (RAR-beta(2)) expression is suppressed in oral premalignant lesions and head and neck squamous cell carcinomas (HNSCCs). This study was conducted to determine whether RAR-beta(2) gene expression in such lesions can be silenced by promoter methylation. RAR-beta(2) methylation was analyzed in DNA samples from 22 pairs of primary HNSCC and adjacent normal epithelium, 124 samples of oral leukoplakia, and 18 HNSCC cell lines using methylation-specific PCR. RAR-beta(2) promoter was methylated in 67, 56, and 53% of HNSCC tumors, HNSCC cell lines, and microdissected oral leukoplakia specimens, respectively. RAR-beta(2) hypermethylation was confirmed by sodium bisulfite-PCR combined with restriction enzyme digestion analysis and by random cloning and sequencing of bisulfite-treated DNA isolates. Significantly higher RAR-beta(2) hypermethylation levels were found in tumor tissue compared with adjacent normal tissue (P = 0.002). RAR-beta(2) methylation in the cell lines was correlated with loss of RAR-beta(2) expression (P = 0.013) and inversely related to the presence of mutated p53 (P = 0.025). The demethylating agent 5-aza-2'-deoxycytidine (5-aza-CdR) restored RAR-beta(2) inducibility by all-trans-retinoic acid (ATRA) in some of the cell lines, which posses a methylated RAR-beta(2) promoter. In some cell lines, this effect was associated with increased growth inhibition after combined treatment with 5-aza-CdR and ATRA. RAR-beta(2) silencing by methylation is an early event in head and neck carcinogenesis; 5-Aza-CdR can restore RAR-beta(2) inducibility by ATRA in most cell lines, and the combination of 5-aza-CdR and ATRA is more effective in growth inhibition than single agents.