Two determinants of hepatitis B surface Ag (HBsAg), identified by mAb raised against polypeptide components, were characterized immunochemically. One was expressed on HBsAg irrespective of the four major subtypes, i.e., adw, adr, ayw, and ayr, whereas the other was subtypic but not identical to any of d, y, w, and r determinants. The common determinant was generated by a synthetic pentadecapeptide with a sequence of Thr-Thr-Ser-Thr-Gly-Pro-Cys-Lys-Thr-Cys-Thr-Ile-Pro-Ala-Gln representing amino acids 115-129 of the S gene product, and detected invariably in 366 HBsAg samples in sera from asymptomatic carriers in Japan. The activity of the S gene product, as well as the peptide (115-129), to bind with the mAb was not affected by alkylation alone, but was completely lost after reductive alkylation. The antigenic activity was lost when the S gene product was severed between Lys122 and Thr123 by trypsin. A microconformation maintained by the -Cys121-Cys124 bond, therefore, would be required for the common determinant. The other mAb identified an epitope of HBsAg that was mimicked by a synthetic tetradecapeptide with a sequence of Thr-Cys-Thr-Ile-Pro-Ala-Gln-Gly-Thr-Ser-Met-Phe-Pro-Ser, representing amino acids 123-136 of the S gene product. Among 16 HBsAg samples with known S gene sequences, 5 with Ile126 possessed this subtypic determinant, but the remaining 11 with Thr126 did not. The 5 hepatitis B virus genomes encoding the subtypic determinant differed less than 5.6% from each other in the entire nucleotide sequence, but by 8.0% or more from any of the other 11 genomes without the capacity to encode it.