The major surface protein of HBV, the 226-amino-acid HBsAg, is encoded in the 3′ proximal segment of the preS-S gene of 389 codons. To identify gene products from the 5′ proximal preS sequence, DNA fragments from the preS region were expresed in Escherichia coli as fusion proteins. Antisera prepared against these fusions were used to screen serum proteins of HBV-infected individuals, and found to react specifically with the two large HBV surface proteins of 39 and 42 kDa. The presence of these proteins could be correlated with acute HBV infection. Analysis by Western blotting using the preS sequence-specific antisera and HBV particles separated into spheres, filaments, and Dane particles confirmed that these proteins were associated with the native virus. Dane particles containing active DNA polymerase could be immune precipitated by the preS-specific antibodies, showing that the preS-coded part of these surface proteins is located on the surface of the virion.