Tumor suppressor genes can become inactivated in cancer via hypermethylation of their promoter. The retinoic acid receptor beta (RARbeta) gene is expressed from two distinct promoters, both of which have CpG islands. RARbeta1 is expressed primarily during embryogenesis, whereas RARbeta2 is expressed in adult tissues and hypermethylated in a number of cancer cells. We used combined bisulfite restriction analysis to evaluate their methylation in colorectal mucosa and tumors. Methylation of RARbeta1 was detected, with a mean of 2% in normal colon tissues in young subjects (< 32 years), and 16% in older subjects (> 75 years) (P < 0.001). Using paired normal/tumor tissue samples, we found higher mean methylation rate in tumors than in adjacent normal tissue (mean, 46% versus 16%; P < 0.001) and hypermethylation of RARbeta1 in all eight cell lines examined. By RT-PCR, RARbeta1 was not expressed in normal adult colon tissues and its expression could not be efficiently activated in most cell lines by the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR). RARbeta2 methylation was also observed in normal colon tissues and was lower in young individuals than in older ones (mean, 11% versus 23%; P < 0.05). Among paired samples, RARbeta2 methylation was higher in tumor tissue than in normal tissue in 14 cases, vice versa in 7 cases, and equal in 6 cases. All eight cell lines were hypermethylated and did not express RARbeta2, but RARbeta2 expression could be reactivated easily by 5-Aza-CdR. We suggest that the embryonic RARbeta1 isoform is readily hypermethylated in aging colon mucosa and all colorectal cancers because of its lack of expression in normal tissues. The adult RARbeta2 isoform also shows age-related methylation in normal tissues but more variable methylation in colorectal cancer, perhaps because its expression offers continued protection against methylation or its silencing does not provide a selective advantage in the early stages of the disease.