Hepatocellular carcinoma (HCC) is one of the most common cancers in the world and is particularly prevalent in Henan, China. The objective of this study was to analyze the frequency and specificity of autoantibodies associated with HCC in Henan. In the present study, 137 sera from HCC patients, 77 sera from other liver diseases, and 30 sera from normal human individuals were examined for autoantibodies using immunohistochemistry, Western blotting, and immunoprecipitation assays. Autoantibodies were detected in 80 of 137 (58.4%) HCC sera. Antinucleolar antibodies were seen more frequently in HCC compared to other liver diseases (9.5% vs. 1.3%, P < 0.05). Two nucleolar proteins-fibrillarin and NOR-90/ hUBF-were identified as autoantigens. The frequency of autoantibodies in HCC sera with known hepatitis C virus (HCV) infection was significantly higher than that in sera without HCV infection (84.2% vs. 57.7%, P < 0.01). Another interesting finding was that autoantibodies to a 90-kDa cytoplasmic antigen were found in 21% of HCC patients. This is the first report on the frequency and specificity of autoantibodies in sera from Chinese patients with HCC. The data support that autoimmune responses to certain cellular proteins may be a by-product in the transformation to HCC, and further studies of novel targeted autoantigens in Chinese HCC may provide insights into how these proteins might be involved in malignancy.