We combined the nested polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) for genotypic identification of Rickettsia tsutsugamushi. Four primers were selected from the DNA sequence of the gene encoding a 56-kD serotype-specific antigen of the Karp strain. Nested PCR produced rickettsia-specific products of approximately 0.6 kb in the amplification of DNA prepared from three reference strains (Gilliam, Karp, and Kato) and two prototype strains (Irie and Hirano) prevalent in the Miyazaki Prefecture of Japan. When the nested PCR products obtained from these five strains were digested with Hha I, profiles specific to each strain were generated. Fourteen of 17 DNA samples of peripheral blood mononuclear cells from patients with scrub typhus tested positive in the nested-PCR, providing a rickettsia-specific band. The serotype of infected rickettsia of 10 patients were diagnosed as Irie and those of four patients were diagnosed as Hirano by indirect immunofluorescence methods. The fragment profiles of the PCR products of these 14 patients after digestion with Hha I corresponded closely with those serotypes. However, the PCR products from two of four samples, which were similar to Hirano strain by a serologic method and by the pattern of digestion with Hha I, produced different RFLP profiles upon further digestion with Hinf I and Alu I. These results may suggest that genetic variation exists within serotypes. Genotypic identification of R. tsutsugamushi by means of PCR-RFLP using three restriction enzymes is apparently useful.