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Aberrant methylation of CpG islands can be a good molecular marker for identifying genes inactivated in cancer. We found the proapoptotic gene HRK to be a target for hypermethylation in human cancers and examined the role of such methylation in silencing the gene's expression.Methylation of HRK was evaluated by bisulfite-PCR and bisulfite sequencing in a group of colorectal and gastric cancer cell lines and primary cancers. Gene expression and histone acetylation were examined by reverse transcription-PCR and chromatin immunoprecipitation analyses, respectively. Apoptosis of cancer cells after treatment with a DNA methyltransferase inhibitor and/or histone deacetylase inhibitor was examined with fluorescence-activated cell-sorting analysis.The region around the HRK transcription start site was methylated in 36% of colorectal and 32% of gastric cancer cell lines and was closely associated with loss of expression in those cell types. HRK expression was restored by treatment with a methyltransferase inhibitor, 5-aza-deoxycytidine, and enhanced further by addition of histone deacetylase inhibitor trichostatin A or depsipeptide. Such restoration of HRK expression was well correlated with induction of apoptosis and enhancement of Adriamycin-induced apoptosis. Expression of other proapoptotic genes, including BAX, BAD, BID, and PUMA, was unaffected by treatment with 5-aza-deoxycytidine. Aberrant methylation of HRK was also frequently detected in primary colorectal cancers that showed methylation of multiple genes, including p16INK4A and hMLH1, and was associated with wild-type p53.HRK methylation can be a useful molecular target for cancer therapy in a subset of colorectal and gastric cancers.