It was recently reported that RUNX3 gene expression is significantly downregulated in human gastric cancer cells due to hypermethylation of its promoter region or hemizygous deletion (Cell, 109, 2002). To verify the genetic alterations and methylation status of the RUNX3 gene in colorectal carcinogenesis, we analysed for mutations, loss of heterozygosity (LOH), and RUNX3 gene promoter hypermethylation, in 32 colorectal cancer cell lines. RT–PCR analysis showed undetectable or low RUNX3 expression in 16 cell lines, and no mutations were found in the RUNX3 gene by PCR-SSCP analysis. Of these 16 cell lines, hypermethylation of the RUNX3 promoter was confirmed in 12. The following observations were made:(i) RUNX3 was re-expressed after 5-aza-2′-deoxycytidine treatment,(ii) the RUNX3 promoter was found to be methylated by MS-PCR, and (iii) hypermethylation of the RUNX3 promoter was confirmed by direct sequencing analysis after sodium bisulfite modification in the above 12 cell lines. RUNX3 was neither methylated nor expressed in four cell lines. Of these four, microsatellite instability (MSI) at the RUNX3 locus was found in three, SNU-61 (D1S246), SNU-769A, and SNU-769B (D1S199). This study suggests that transcriptional repression of RUNX3 is caused by promoter hypermethylation of the RUNX3 CpG island in colorectal cancer cell lines, and the results of these experiments may contribute to an understanding of the role of RUNX3 inactivation in the pathogenesis of colorectal cancers.