The SSX genes are members of the cancer-testis (CT) antigen family. SSX2, the prototype SSX gene, was found by serological analysis of antigens by recombinant expression cloning (SEREX). Since little is known about SSX expression in gynecological malignancies, we investigated SSX mRNA expression in 115 gynecological cancer specimens and 25 normal control samples by RT-PCR. We also tested the humoral immune response to SSX2 and SSX4 using recombinant proteins. We found relatively high SSX4 mRNA expression in endometrial cancer (24%), ovarian cancer (13%), and cervical cancer (20%). In contrast, SSX1 and SSX2 mRNA expression was detected in not more than 4% of the gynecological cancer specimens analysed. No SSX mRNA expression was found in 25 normal specimens. Two gynecological cancer patients had antibodies against SSX4, whereas no antibody reactivity to SSX4 was found in the sera of 40 normal individuals. This suggests that SSX4 is a potential target for cancer vaccines in gynecological cancer patients.