The 55 codons upstream of the gene sequence encoding the hepatitis B surface antigen (HBsAg) are called the pre-S(2) region. It has been proposed that polypeptides of high molecular weight that contain the pre-S(2) region should be included in future hepatitis B virus (HBV) vaccines. The pre-S(2) region and the S gene product [25 kilodalton (kD)] together compose a polypeptide of high molecular weight (33 kD). As an initial attempt to determine the relevance of the 33-kD polypeptide to development of an HBV vaccine, the murine immune response to pre-S(2)-encoded determinants as compared to S-encoded determinants on the same polypeptide was examined. The results indicate (i) the pre-S(2) region is significantly more immunogenic than the S region of HBsAg, (ii) the 26 amino acid residues at the NH2-terminus of the 33-kD polypeptide represent a dominant antibody binding site on the pre-S(2) region, (iii) the immune response to the pre-S(2) region is regulated by H-2-linked genes distinct from those that regulate the response to the S region, and (iv) immunization of an S region nonresponder strain with HBV envelope particles that contain both the pre-S(2) and S regions can circumvent nonresponsiveness to the S region.