Sera of patients from China with hepatocellular carcinoma (HCC) were tested for the presence of HBc/HBe- and HBx antibodies by immunoblotting using recombinant MS2 or beta gal fusion proteins as substrate. Antibodies against HBx were detected in four out of 68 HBsAg positive and in one out of three HBsAg negative sera, antibodies against HBc/HBe in 52 and two serum samples, respectively. Competition experiments in which sera were preincubated with individual viral proteins synthesized in E. coli were carried out to demonstrate the specificity of signals obtained in immunoblot analyses. In the five anti-HBx positive sera, the antibody titer against X fusion protein was higher than against core fusion protein and in one of these sera anti-x activity could be demonstrated even at a serum dilution of 1:50,000. These data indicate that X antibodies occur rarely in Chinese patients and are not serodiagnostic for HCC. The high titer of X antibodies in some patients shows that the X protein can be highly immunogenic in vivo. Induction of antibody formation may be triggered by X protein expressed from integrated viral DNA.