In this study, we identified 53 aberrantly hypermethylated DNA sequences in adult T-cell leukemia (ATL) cells using methylated CpG island amplification/representational difference analysis method. We also observed a proportionate increase in the methylation density of these regions with disease progression. Seven genes, which were expressed in normal T cells, but suppressed in ATL cells, were identified near the hypermethylated regions. Among these silenced genes, Kruppel-like factor 4 (KLF4) gene is a cell cycle regulator and early growth response 3 (EGR3) gene is a critical transcriptional factor for induction of Fas ligand (FasL) expression. Treatment with 5-aza-2'-deoxycytidine resulted in the recovery of their transcription, indicating that their silencing might be associated with DNA hypermethylation. To study their functions in ATL cells, we transfected recombinant adenovirus vectors expressing KLF4 and EGR3 genes. Expression of KLF4 induced apoptosis of ATL cells whereas enforced expression of EGR3 induced the expression of FasL gene, resulting in apoptosis. Thus, suppressed expression of EGR3 enabled ATL cells to escape from activation-induced cell death mediated by FasL. Our results showed that the methylated CpG island amplification/representational difference analysis method allowed the isolation of hypermethylated DNA regions specific to leukemic cells and thus shed light on the roles of DNA methylation in leukemogenesis.