The expression of a previously unidentified gene product, encoded by the hepatitis B virus (HBV) genome, has been achieved with a recombinant SV40 expression vector. Antibodies against synthetic peptides representing defined regions of this protein were used to screen cells infected with recombinant virus as well as tissues naturally infected with HBV. A 24,000-dalton protein (p24) was detected in cells infected with recombinant virus and a 28,000-dalton protein (p28) was detected in tissues infected with HBV. The peptides or recombinant-derived protein were used as antigens to screen sera from individuals infected with HBV. Specific antibodies were detected predominantly in sera from patients with hepatocellular carcinoma. The presence of p28 in tissues infected with HBV and the appearance of specific antibodies in infectious sera establish the existence of an additional marker for HBV infection.