The heterogeneous antigenic determinants of Hepatitis B antigen (HB Ag) from 310 individuals were compared by using a micro-Ouchterlony immunodiffusion technique. Reference antisera were made in rabbits immunized with a purified HB Ag with ay determinants from an American with hepatitis and another purified HB Ag with ad determinants from a Thai blood donor. The immunizing antigens were prepared by a combination of equilibrium centrifugation in cesium chloride and rate zonal centrifugation in sucrose gradients. Three immunodiffusion patterns of antigen reactivity were observed: pattern 1 antigens showed identity to the reference ay HB Ag; pattern 2 antigens showed identity to the reference and HB Ag and pattern 3 antigens showed partial identity to both reference antigens. Further study after absorbing the antisera with heterologous antigens indicated that the reference antisera were distinguishing two additional antigenic determinants, designated w and r. Preliminary data suggest w is more common that r on HB Ag in the United States, but r is most common in Thailand.