Since 1975, there has been an increase in the number of patients with tsutsugamushi disease in Japan, and marked antigenic heterogeneity has been found among newly isolated strains of Rickettsia tsutsugamushi. For antigenic analysis of these strains, we produced monoclonal antibodies against the Irie strain isolated in 1971, and the Hirano and Shimokoshi strains isolated in 1980. In all, 34 monoclonal antibodies were produced and their reactivities were determined by the immunofluorescent antibody test. The serological reactivity of the antibodies against these three strains and classic representative strains (Gilliam, Karp and Kato) showed varied reactive characteristics, i.e., serotype-specific, species-specific and intermediate reactivities. It was revealed that these strains are antigenically different from the classic ones. Moreover, by using the serotype-specific monoclonal antibodies, nine strains newly isolated in Miyazaki Prefecture were classified into the Irie and the Hirano types. The antigenicity of the Shimokoshi strain differed from those of the other strains used in this study. From these results, the strains of R. tsutsugamushi used in this study fell into six serotypes including the classic strains. SDS-PAGE and immunoblotting were performed to determine the molecular sizes of the antigenic polypeptides. The results revealed that the serotype-specific antigens belong to the 60-kDa class whereas the species-specific antigens belong to the 61-kDa, 60-kDa or 44-kDa class.