Cancer-testis (CT) genes are expressed in a variety of human cancers, but not in normal tissues except for testis, and represent promising targets for immunotherapy and gene therapy. We investigated the expression of 10 CT genes (MAGE-1, MAGE-3, MAGE-4, GAGE, NY-ESO-1, SSX-1, HOM-MEL-40/SSX-2, SSX-4, HOM-TES-14/SCP-1, and HOM-TES-85) in 21 hepatocellular carcinoma (HCC) biopsy specimens. The most frequently expressed CT genes were SSX-1 and GAGE, which were found in 8/21 (38%) HCC samples, followed by HOM-TES-14/SCP-1 (6/21 or 29%), MAGE-3 (5/21 or 24%), HOM-TES-85 and MAGE-1 (4/21 or 19% each), whereas SSX-4 and HOM-MEL-40/SSX-2 were only expressed in 2/21 cases each, MAGE-4 in one case, and NY-ESO-1 not at all. Of the 21 HCC cases investigated, only four did not express any of the CT genes tested, 17 (81%) expressed at least one, 9 (43%) coexpressed two, four (19%) coexpressed four, three (14%) coexpressed five and one coexpressed 8 of the 10 CT genes tested. We conclude that a majority of HCC cases might be amenable to specific immunotherapeutic interventions. However, the identification of additional tumor-specific antigens with a frequent expression in HCCs is warranted to develop widely applicable, polyvalent HCC vaccines.