We used the polymerase chain reaction (PCR) for the diagnosis of an acute rickettsial infection. A primer pair derived from the 17-kDa antigen sequence of Rickettsia rickettsii amplified specifically a 434-bp DNA fragment from the genome of Rocky Mountain spotted fever and endemic and epidemic typhus. The assay was able to detect as few as 30 rickettsiae. Detection of PCR-amplified DNA with a digoxigenin-labeled DNA probe confirmed an acute human infection with Rickettsia prowazekii.