The effects of retinol (vitamin A) depend on its transport and binding to nuclear receptors. The cellular retinol-binding protein 1 (CRBP1) and the retinoic acid receptor beta2 (RARbeta2) are key components of this process. Loss of CRBP1 expression occurs in breast tumors, but the mechanism is not known. We examined whether CpG island hypermethylation of CRBP1 was responsible for its inactivation in cancer cell lines (n = 36) and primary tumors (n = 553) and its relation to RARbeta2 methylation. Hypermethylation of CRBP1 was common in tumors and cancer cell lines, with the highest frequency in lymphoma and gastrointestinal carcinomas. Hypermethylation correlated with loss of CRBP1 mRNA, and in vitro treatment with the demethylating agent 5-aza-2'-deoxycytidine reactivated CRBP1 expression. CRBP1 methylation appeared in premalignant lesions and frequently occurred with RARbeta2 hypermethylation in the same tumors. Finally, we observed that a higher dietary retinol intake was associated with reduced frequencies of methylation of both genes. Epigenetic disruption of CRBP1 is a common event in human cancer that may have important implications for cancer prevention and treatment using retinoids.