CDX2 (caudal type homeobox transcription factor 2) is a homeobox protein, which is expressed in intestinal epithelium. CDX2 has been considered to play a role as a tumor suppressor gene in colorectal cancer because its expression is lacking in colorectal carcinomas, but preserved in adenomas. The point mutations have been found to account for loss of CDX2 expression, but the main mechanism responsible for CDX2 gene inactivation is less understood. We analyzed methylation and expression status of CDX2 in colorectal cancer cell lines and primary tumors. There are two CpG-rich sites in promoter region of CDX2 gene, -1570 to -1200 and -220 to +880. By COBRA (combined bisulfite restriction analysis) assay, the upper CpG-rich site was heavily methylated in all cell lines, but the lower CpG-rich site was methylated in limited cell lines. Bisulfite sequencing analysis and RT-PCR revealed that methylation of the lower CpG site was associated with down-regulation of CDX2. In addition, gene expression was restored in COLO201, a methylated cell line with 5-aza-2'-deoxycytidine, confirming that methylation caused gene down-regulation. We also examined CDX2 promoter methylation of primary tumors by MSP (methylated-allele specific PCR) assay and found that nearly 40% of cases have a methylated CDX2 gene. Our results demonstrate that CDX2 methylation is frequently present in colorectal cancers and may play a key role in inactivating CDX2 expression.