The HCA587 gene, identified by serological analysis of recombinant cDNA expression library (SEREX) from a hepatocellular carcinoma (HCC) patient, encodes a new member of cancer-testis antigens. HCA587 mRNA expression in normal tissues and cancers has been previously reported. To estimate its immunogenicity to induce immune response, it is essential to analyze HCA587 expression at the protein level. In this study anti-HCA587 polyclonal antibody, termed "TC-1," was generated, and the expression of HCA587 protein was assessed by immunohistochemical staining in a panel of normal and tumor tissue sections. No HCA587 protein was shown in normal tissues except germ cells in testis and Purkinji cells in cerebellum. In HCC specimens the HCA587 protein was expressed in 37.1% (26 of 70) samples. The expressed protein was either located in the cytoplasm or nucleus depending on the individual samples. More importantly, there appears to be correlation between the tumor differentiation of HCC and HCA587 protein expression, ie, the lower differentiation, the higher percentage of protein expression. Coincidentally, seroreactivity showed that the Ab specific to recombinant HCA587 protein was detected only in the sera of three patients with poorly differentiated HCCs. HCA587 antigen was also expressed in different proportions in melanoma, lymphoma, pancreatic cancer, and lung cancer.