Monoclonal antibodies with restricted specificity were used in a modified commercial enzyme immunoassay for detection of HBsAg to subtype HBsAg in sera from 122 Southeast Asian refugees entering the United States, 62 inmates of a correctional facility, and 19 homosexual men. This method was able to classify HBsAg as aywl-2, ayw3, ayw4, ayr, adw2, adw4, or adr. The HBsAg subtype was identified in 183 (90.1%) of the serum samples, but the serum HBsAg concentration was too low to determine the subtype for the 20 (9.9%) remaining samples. Among the Southeast Asian refugees, aywl-2 was demonstrated in 35 (33.0%) of the subtyped serum samples, the adw2 subtype was identified in 33 (31.1%) sera, adr was detected in 37 (34.9%) sera, and the adw 4 subtype as found in 1 (0.9%). The most common subtypes in Vietnam, Laos, and Cambodia were aywl-2, adw2, and adr, respectively. In prison inmates, the ayw3 subtype accounted for 31 (52.5%) of the subtyped serum samples, an ayw2 variant and the adw2 subtype were each found in 13 (22.0%) sera, and the aywl-2 subtype was detected in 2 (3.4%) sera. Many of these inmates admitted intravenous drug use. Among homosexual men, the adw2 subtype was identified in 16 (88.9%) of the subtyped serum samples and the ayw3 subtype was detected in 2 (11.1%) sera. This subtyping method can distinguish between most of the nine major HBsAg subtypes and can be easily performed with these monoclonal antibodies and commercially available reagents.