The methylation inhibitor 5-Aza-2′-deoxycytidine (decitabine) has therapeutic efficacy in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Using microarray analysis, we investigated global changes in gene expression after decitabine treatment in AML. In the AML cell line OCI-AML2, decitabine induced the expression of 81 out of 22 000 genes; 96 genes were downregulated (X2-fold change in expression). RT-PCR analysis of 10 randomly selected genes confirmed the changes of expression in AML cells. Similar results were obtained with primary AML and MDS cells after treatment with decitabine ex vivo and in vivo, respectively. In contrast, significantly fewer changes in gene expression and cytotoxicity were detected in normal peripheral blood mononuclear and bone marrow cells or transformed epithelial cells treated with decitabine. Interestingly, only 50.6% of the induced genes contain putative CpG islands in the 5′ region. To further investigate the significance of promoter methylation in the induced genes, we analyzed the actual methylation status of randomly selected decitabine-inducible genes. We detected hypermethylation exclusively in the 5′ region of the myeloperoxidase (MPO) gene. DNA methylation inversely correlated with MPO expression in newly diagnosed untreated AML patients (P<0.004). In contrast, all other analyzed decitabine-inducible genes revealed no CpG methylation in the promoter region, suggesting a methylation-independent effect of decitabine.