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Forty-nine cases of murine typhus were diagnosed in recent years in residents of several communities around the city of Chalkis, the capital of the Prefecture of Evia. (Euboea) Evia is an island connected to central mainland Greece by a bridge. To investigate the endemicity of murine typhus in this area, 226 fleas (Xenopsylla cheopis) and blood samples were collected from 53 rats (Rattus norvegicus) trapped in this area. The polymerase chain reaction followed by restriction fragment length polymorphism analysis (PCR-RFLP) was used to detect and identify Rickettsia typhi, the etiologic agent murine typhus, in the rat blood samples (buffy coat cells) as well as in their fleas. An indirect immunofluorescent antibody (IFA) assay was performed to detect antibodies against R. typhi in rat serum samples. The presence of R. typhi in both fleas and rat blood samples was demonstrated. The frequency of infection for X. cheopis was 4%, while 18% of the rats had buffy coat cells infected, and 92% of the rat sera tested by IFA were positive for anti-R. typhi antibodies. The present work is the first successful application of PCR-RFLP in a field study of naturally infected rats and their fleas in Europe.