We developed a nested polymerase chain reaction (PCR) method to detect Rickettsia tsutsugamushi (R. tsutsugamushi) DNA and determined its sensitivity. Primers were selected from the DNA sequence of the 58-kDa group-specific antigen gene of the Karp strain. The target sequence of rickettsial DNA was detectable as the band corresponding to 88 bp in 1.0 microgram of the DNA extracted from BS-C-1 cells infected with R. tsutsugamushi. Rickettsia-specific bands were observed not only for the homologous Karp strain, but also for four heterologous strains: two other reference strains (Gilliam and Kato) and two prototype strains prevalent in Miyazaki district (Irie and Hirano). The minimum copy number detectable by this method was estimated to be five rickettsiae. All of nine peripheral blood mononuclear cell samples from patients with tsutsugamushi disease who were seen 2-11 days after disease onset tested positive for rickettsial DNA. The PCR assay method presented here could be a specific diagnostic tool for tsutsugamushi disease, especially in its early acute stage.