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The retinoic acid receptor β (RAR β), a putative tumor suppressor gene, has been reported to be poorly expressed in breast cancer. In this report using the methylation-specific PCR reaction we observed DNA methylation in the promoter region of RAR β in several primary breast tumors. DNA sequence analysis showed that the positions of 5-methyl-cytosine in the RARβ promoter region was almost identical to that reported previously by our laboratory for human DLD-1 colon carcinoma cells (Anti-Cancer Drugs 1998; 9: 743). Several other cancer-related genes have been also reported to be silenced by DNA methylation, including the p16 tumor suppressor gene, E-cadherin, an invasion suppressor gene and the estrogen receptor gene in breast cancer cell lines. Since breast cancer cells have several potential target genes for the DNA methylation inhibitor, 5-aza-2‘-deoxycytidine (5-Aza-CdR), we investigated the in vitro antineoplastic activity of this analog on the human breast cancer cell line MDA-MB-231. We report that 5-Aza-CdR is a potent growth inhibitor and a potent cytotoxic agent against the breast carcinoma cells. These results suggest that 5-Aza-CdR may be an interesting agent to investigate in patients with breast cancer resistant to conventional chemotherapy.