The broad range of expression of cancer-testis antigens in various tumor types makes the proteins encoded by human MAGE gene family promising targets for anticancer immunotherapy. However, a major drawback is their heterogeneous expression. In the current study, we have examined the influence of the DNA methylase inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) together with the histone deacetylase inhibitor trichostatin A on the expression of MAGE-A1, -A2, -A3, and -A12 genes in different cell lines. Reverse transcription-PCR, Western blot analyses, and immunocytochemical staining show that trichostatin A was able to significantly up-regulate 5-aza-CdR-induced MAGE gene expression. Transient transfection assays with methylated reporter plasmids containing promoter fragments of the different MAGE genes show that trichostatin A was able to overcome gene silencing. In addition, the methylation status of the MAGE promoters was assessed by sodium bisulfite mapping in the various cell lines before and after stimulation with 5-aza-CdR and/or trichostatin A. In contrast to the methylation patterns, which clearly correlated with the basal MAGE RNA transcripts, up-regulation of the MAGE-A mediated by both agents only resulted in a reduction in promoter methylation ranging between 1% and 19%. In conclusion, our data show for the first time that not only hypermethylation but also histone deacetylation is responsible for the mechanism underlying MAGE gene silencing.