Lack of information regarding the presence of native albumin polymer in serum and its structural similarity to the one produced by glutaraldehyde treatment casts doubt on the postulate that hepatitis B virus attachment to hepatocytes is mediated through polymerized albumin. We used a sandwich enzyme-linked immunosorbent assay with murine monoclonal antibodies raised against glutaraldehyde-polymerized albumin to detect native albumin polymer in human serum and its cross-reactivity with other albumin polymers. Presence of polymerized albumin receptor on the HepG2 cell was studied by radioreceptor assay. Purified hepatitis B virus and synthetic peptide analogous to part of pre-S2 sequence (120-145) were used to study polymerized albumin-dependent attachment of the virus to HepG2 cells. Antibodies raised against pre-S2 peptide were used to inhibit the pre-S2 and hepatitis B virus attachment to HepG2 cells. Glutaraldehyde-treated polymerized albumin was found to be immunologically cross-reactive with native albumin polymer. Its levels were found to be significantly raised in sera of patients with liver diseases. Polymerized albumin has specific saturable receptor on HepG2 cells with two classes of binding sites of different equilibrium dissociation constant (Kd, = (16 * 9.6)pmolL and Kd, = (1,019 * 172)pmolL. Albumin monomer was unable to compete for the polymerized albumin receptor sites on HepG2 cells. Antipre-S2 antibodies inhibit hepatitis B virus and pre-S2 binding to hepatocyte by 40% and 70%, respectively. Added extraneous polymerized albumin and the antibody against it did not interfere with virus attachment to HepG2 cells. (HEPATOLOGY 1991;13:134-142.) For initiation of viral infection, attachment and entry of the virus to the susceptible host cell is of extreme importance. Two types of receptors have been proposed for attachment of HBV to hepatocytes, one for pre-S1 region of HBsAg and the other for pre-S2 region, possibly mediated through polymerized albumin (PHSA)