Abstract The large (L) surface glycoprotein of hepatitis B virus is an important component of the virion envelope derived from translation initiation at the 5′ end of the PreS1 domain of the surface antigen open reading frame. Since key roles in virion assembly and infectivity have been postulated for this protein, further understanding of its structure and topology is important. To this end we have mapped the epitopes recognized by a panel of monoclonal antibodies specific for this polypeptide by examining their reactivity with a series of deletion mutants of the PreS1 region expressed in cultured cells. On the basis of this and other techniques, the antibodies fall into two groups mapping to two distinct epitopes spanning residues 27–35 and 72–78, respectively. Immunoprecipitation studies indicate that both regions are exposed on the surface of HBV-encoded particles.