HBsAg subtypes were defined by means of adsorbed polyclonal antisera; however, HBsAg subtyping is currently usually carried out with monoclonal antibodies (Mab). We developed a complementary subtyping method based on the polymerase chain reaction. Reference samples belonging to all known HBsAg subtypes could be detected and grouped into four different categories (ayw1/ayw4/ayr, ayw2/ayw3, adw2/adrq+/adrq-, adw4). Thirteen HBsAg-positive serum samples previously subtyped as ad by means of monoclonal antibodies fell into the adw2/adrq+/adrq- group, as well as 13 ay samples into the ayw2/ayw3 group. These results could be confirmed by means of reference polyclonal antisera in nine ad cases (all adw2) and in seven ay cases (all ayw3); the remaining seven were below the detection limit of the polyclonal assay. Four samples which were not recognized by any of the d/y subtype-specific Mab were shown to contain ayw2/ayw3 sequences. Only one contained sufficient HBsAg to be confirmed as ayw3 by means of reference antisera. Three of five sera showing simultaneous reactivity both for d and y-specific Mab were classified as adw4 by PCR, as was one by reference polyclonal antisera. The y-specific monoclonal antibody cross-reacted with the adw4 subtype. Single adw2 sequences were amplified in one of the remaining two cases, as well as single ayw2/ayw3 sequences in the other, suggesting that they showed true coexistence of two strains of different subtype, only one of which was in active replication state. It is concluded that the method described is useful in the solution of some undefined cases obtained with the monoclonal-based assays. © 1994 W iley-Liss, Inc.