OBJECTIVE: The p14(ARF) and p16(INK4A) tumor suppressor genes are commonly inactivated by aberrant methylation of their promoter regions in human colon cancer. The methyl-CpG-binding domain protein MBD2 is physically associated with the methylated promoters of the p14(ARF) and p16(INK4A) genes in specific tumor cell lines. Moreover, deficiency of MBD2 strongly inhibits intestinal tumorigenesis in the Min mouse, raising the possibility that the protein might be involved in transcriptional repression of methylated tumor suppressor genes. The aim of this study was to evaluate the role of MBD2 in the silencing of p14(ARF) and p16(INK4A) in cancer. METHODS: The MBD2 protein was stably knocked down by RNA interference in RKO, a colon cancer cell line in which both p14(ARF) and p16(INK4A) are silenced by methylation. RESULTS: We demonstrate here that MBD2 associates with the methylated promoter of the p14(ARF) gene in the RKO colon cancer cell line. Depletion of MBD2 by RNAi leads to selective upregulation of the p14(ARF) but not the p16(INK4A) gene transcript. In addition, p14(ARF) repression can be restored by expressing mouse MBD2 protein in MBD2-deficient RKO cells. CONCLUSION: These findings implicate MBD2 in transcriptional repression of the methylated p14(ARF) tumor suppressor gene and suggest that repression by MBD2 selectively affects a subset of methylated promoters.