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We previously reported that 5-aza-2’-deoxycytldlne (5-Aza- CdR) In combination with a\\-trans retinoic acid (ATRA) produced a synergistic antineoplastic effect on DLD-1 colon carcinoma cells. We also observed that 5-Aza-CdR, a potent inhibitor of DNA methylation, increased the expression of retinoic acid receptor (RAR)-β). Methylation of cytosine in the promoter-first exon region of genes has been reported to silence their expression. In an attempt to clarify the mechanism responsible for the activation of the RAR-β gene by 5-Aza-CdR in DLD-1 colon carcinoma cells, we investigated its methylation state by Southern blotting. Our results indicate that DNA hypermethylation of the RAR β gene, a putative tumor suppressor gene, may be the mechanism of silencing its expression in these tumor cells. We also reported that a different schedule of 5-Aza-CdR and ATRA produced a synergistic antineoplastic effect on the colon carcinoma cells.