Vaccination-based therapy of melanoma has so far mainly focused on monovalent approaches using either melanoma differentiation antigens or cancer/testis antigens. To study the complementarity of expression from these two families of antigens recognized by T-cells, we screened 47 metastatic lesions of cutaneous melanoma for the expression of three melanoma differentiation antigens and eight cancer/testis antigens using reverse transcription-polymerase chain reaction (RT-PCR). The melanoma differentiation antigens were expressed in a somewhat higher percentage of lesions (94% positive for at least one marker) than the cancer/testis antigens (91% positive for at least one marker). Nearly all the melanoma metastases (98%) expressed at least one of the markers tested. One melanoma metastasis was negative for all the markers. Two out of 47 lesions did not express any of the three differentiation markers but expressed one or more of the cancer/testis antigens, indicating some additional potential for these antigens compared with the melanoma differentiation antigens. Therefore, we conclude that polyvalent immunotherapy using multiple epitopes from both families of antigens might increase the eligibility of melanoma patients and the efficacy of the treatment.