The role of retinoic acid receptor beta(RARbeta), a putative tumor suppressor gene, in the development of colon malignancy still remains to be clarified. We reported previously that the expression of RARbeta in DLD-1 human colon adenocarcinoma cells was silenced by DNA methylation at the level of the promoter region (Anti-Cancer Drugs 1997; 8: 56). In addition, we observed that RARbeta expression could be activated by the hypomethylating action of 5-aza-2'-deoxycytidine (5-Aza-CdR). In this report we have identified, by sequencing of bisulfite-modified DNA of DLD-1 colon tumor cells, the specific 5-methylcytosine positions in the region of -46 to +251 bp from the transcription start site of RARbeta2. We observed that 5-Aza-CdR treatment demethylated these specific sites. Based on this sequence data, specific primers for the methylation-specific PCR (MSP) assay were designed to discriminate methylated from unmethylated CpG sites in the promoter region of RARbeta. This assay confirmed the changes in the methylation status of the RARbeta gene in DLD-1 colon tumor cells before and after treatment with 5-Aza-CdR. The methylation status of the promoter region of the RARbeta gene was also examined in primary human colon adenocarcinomas using the MSP assay. Six of the 14 colon tumor samples showed signs of hypermethylation of this gene. The MSP assay for RARbeta may be a useful tool to clarify the role of DNA methylation for this gene in colon tumorigenesis.