Recently, p53 gene aberrations have been recognized as a relevant factor in hepatocarcinogenesis, in tumors from both high-risk and low-risk areas. Because p53 gene mutations typically result in increased p53 levels in tumor cells, this cellular protein might become immunogenic during tumor development. To test this hypothesis, we have analyzed sera from 80 European patients with hepatocellular carcinoma for the presence of p53 antibodies. For this purpose we developed an immunoblot assay using recombinant p53 as antigen. Sixty-seven sera from patients with different acute and chronic liver diseases were used as controls. In addition, serum alpha-fetoprotein assays were performed. Circulating antibodies against p53 were found in 25% (20 of 80) of the sera from patients with hepatocellular carcinoma but not in various nonmalignant liver diseases. The association of p53 antibodies with malignancy was highly significant (p < 0.00003). In 73.8% (59 of 80) of the hepatocellular carcinoma sera the alpha-fetoprotein levels were elevated. Among the 21 alpha-fetoprotein-negative hepatocellular carcinoma sera, 5 were found to contain p53 antibodies (23.8%). In conclusion, an antibody response against p53 developed in a significant proportion of patients with hepatocellular carcinoma but not in those with nonmalignant liver diseases. Serological testing for p53 antibodies gives the opportunity to identify a subgroup of patients with hepatocellular carcinoma not detected by conventional tests for serum alpha-fetoprotein.