Cancer-testis antigens expressed by different-histotype transformed cells are suitable targets for tumor immunotherapy. However, their heterogeneous expression in neoplastic lesions limits the eligibility of patients for cancer-testis antigen–directed vaccination, and low levels of cancer-testis antigens' expression may impair immune recognition of malignant cells. Because of the primary clinical relevance of cancer-testis antigens' expression in neoplastic tissues, 68 unrelated or sequential metastatic lesions from 56 patients were used to characterize the molecular mechanisms regulating the presence and levels of expression of different cancer-testis antigens of theMAGEfamily (i.e.,MAGE2,3and4) in cutaneous melanoma. Polymerase chain reaction-based methylation analyses showed that methylation status of specific cytosine-guanine dinucleotides in the promoters of investigated cancer-testis antigens correlated with their heterogeneous expression within unrelated metastatic melanoma lesions, and with their homogeneous expression among sequential metastases from three patients with melanoma. Unlike methylated promoters, unmethylated promoters ofMAGE2,3and4genes drove the expression of reporter gene-enhanced green fluorescent protein after transient transfection of cancer-testis antigen–positive Mel 142 melanoma cells. Furthermore, de novo expression ofMAGE3gene induced by the treatment of Mel 195 melanoma cells with the DNA hypomethylating agent 5-aza-2´-deoxycytidine was associated with a 6%–12% demethylation of selected cytosine-guanine dinucleotides in its promoter. Finally, 5-aza-2´-deoxycytidine induced a 16-fold increase ofMAGE3expression in Mel 313 melanoma cells expressing constitutively low levels of the antigen, but did not affect that of Mel 275 melanoma cells expressing high baseline levels ofMAGE3.Overall, these findings identify promoter methylation as a shared mechanism directly regulating the expression of therapeutic cancer-testis antigens in metastatic melanomas, and foresee the clinical use of 5-aza-2´-deoxycytidine to design new chemoimmunotherapeutic strategies in patients with melanoma.