Antibodies to the envelope proteins of duck hepatitis B virus neutralize viral infection in vitro. Using a library of murine monoclonal antibodies (Mabs) against the envelope proteins, we previously identified four neutralizing and two non-neutralizing epitopes on the pre-S region of the large envelope proteins. In this study we report the localization of all but one of these epitopes at the amino acid level. All but 28 nucleotides of the pre-S and S genes were cloned in pUC vectors and expressed in Escherichia coli. All Mabs in this study reacted with the expressed gene products in Western blots. Deletion mutants of the pre-S region were generated and their expressed products tested on Western blots for reactivity with the Mabs. Of the three epitopes involved in neutralization, the epitope found to be immunodominant in convalescent ducks was localized to nine amino acids of the middle portion of the pre-S gene product, while a second epitope was mapped to nine amino acids upstream of the immunodominant epitope and the third epitope to seven amino acids adjacent to the S gene. One of the two non-neutralizing epitopes was located between the two groups of neutralizing epitopes while the other mapped to the same region as one of the neutralizing epitopes. Our data indicate that several regions of the pre-S polypeptide may play a role in neutralization of hepadnaviruses.