Runt domain transcription factors are important targets of TGF-β superfamily proteins and play a crucial role in mammalian development. Three mammalian runt-related genes, RUNX1, RUNX2 and RUNX3, have been described. RUNX3 has been shown to be a putative tumor suppressor gene localized to chromosome 1p36, a region showing frequent loss of heterozygosity events in colon, gastric, breast and ovarian cancers. Because of the important role of TGF-β signaling in the human colon, we hypothesized that RUNX3 may serve as a key tumor suppressor in human colon cancers and colon cancer-derived cell lines. We examined RUNX3 expression and the frequency of RUNX3 promoter hypermethylation in 17 colon cancer cell lines and 91 sporadic colorectal cancers. Semiquantitative analysis of RUNX3 transcripts was performed by RT-PCR and de novo methylation of the RUNX3 promoter was studied by a methylation-specific PCR (MSP) assay. Nineteen of 91 informative tumors (21%) and 11 of 17 (65%) colon cancer cell lines exhibited hypermethylation of the RUNX3 promoter. Interestingly, RUNX3 promoter hypermethylation was more common in tumors exhibiting high frequency of microsatellite instability (MSI-H) (33% of MSI-H vs. 12% of MSI-L/MSS tumors; p = 0.012). Hypermethylation of the RUNX3 promoter correlated with loss of mRNA transcripts in all cell lines. RUNX3 promoter methylation was reversed and its expression restored in SW48 and HCT15 colon cancer cells after treatment with the demethylating agent 5-aza-2′-deoxycytidine, indicating that loss of expression is caused by epigenetic inactivation in colon carcinogenesis. This is the first demonstration of frequent de novo hypermethylation of the RUNX3 promoter in sporadic colon cancers. The significant association of RUNX3 promoter hypermethylation with MSI-H colon cancers suggests that RUNX3 is a novel target of methylation, along with the hMLH1 gene, in the evolution of MSI-H colorectal cancers. © 2004 Wiley-Liss, Inc.