The sequence of the preS1 region of the hepatitis B virus (HBV) envelope (env) proteins contains a dominant binding site for hepatocytes between residues preS21 and preS47. Purified HBV particles (subtype ad) were used as the immunogen to produce specific monoclonal antibodies (McAbs) against three antigenic regions (S, preS2 and preS1) of the HBV env protein. One McAb, F35.25, was found to be specific for the region 32-53 of the preS1 sequence of HBV, which largely overlapped the hepatocyte receptor binding site. The preS1-specific McAb F35.25 reacted with both HBV subtypes, ad and ay, in radioimmunoassays (RIA) and with the large surface proteins, P39 and GP42, as well as with tryptic fragments preS(1-99/103) and preS(1-113) in Western blotting experiments. This McAb F35.25 preferentially recognized, however, the homologous (ad) preS1 sequence in RIA. The ad/ay amino acid substitution within the hepatocyte receptor binding site at position 35 (Gly-Arg) may explain the relative subtype-specificity of F35.25. Finally, the F35.25 epitope was detected in all HBV particles purified from HBeAg-positive human sera, confirming that this preS1 region 32-53 is exposed at the surface of complete virions. Thus, we developed a RIA system allowing us to assess the infectivity of HBV particles by the detection of preS1 sequences associated with the viral hepatocyte receptor. Moreover, it is expected that F35.25 may be a virus-neutralizing antibody by blockage of the attachment of HBV to liver cells.