Recombinant MS2- or beta gal fusion proteins containing parts of hepatitis B virus (HBV) HBx-, HBc-, and HBs-amino acid sequences were expressed in Escherichia coli and were used to screen 96 and 60 serum samples of HBV infected and uninfected patients, respectively, for the corresponding antibodies by immunoblotting. Antibodies against HBx were detected in 20 out of 65 sera of patients with previous resolved HBV-infection, in 3 out of 7 patients with persistent infection, and in 9 out of 24 sera of patients with acute HBV infection. The specificity of the immune reaction was confirmed by competition experiments with MS2- and beta gal-HBx fusion proteins, and by the lack of HBx antibodies in the sera of uninfected patients. Hbs and HBc antibodies were detected less frequently by immunoblotting with recombinant fusion proteins than by a commercial immunoassay. Our results indicate that HBx antibodies are induced early and frequently during HBV infection suggesting that the HBx protein is an early antigenic protein expressed in vivo.