Two-step polymerase chain reaction (nested PCR) method was examined for the diagnosis of scrub typhus. Primers were derived from the type-specific antigen (TSA) gene DNA sequences of Rickettsia tsutsugamushi, Gilliam strain. These primers served to produce rickettsia-specific products in the amplification of template DNA prepared from all serovariants, Gilliam, Karp, Kato, Kawasaki, Kuroki and Shimokoshi strains, and the fragments of product after digestion with several kinds of restriction endonuclease showed the respective patterns to strain in acrylamide or agarose gel electrophoresis. The rickettsia-specific DNAs were also derived, by this nested PCR, by amplifying DNA from patients' bloods and mites from endemic areas, and the serotype of rickettsiae infected to these hosts could be identified from fragment patterns of the amplified products observed after endonuclease treatment. These results indicate that this PCR is sensitive and specific method not only for detection of rickettsial DNA in patient specimens and in mites, but also for the typing of rickettsiae infected to these hosts.