Prevalence data for tick-borne pathogens are used to assess the risk for human health. In this study the presence and identity of Borrelia burgdorferi sensu lato, Ehrlichia, Anaplasma, and Rickettsia species in Bulgarian Ixodes ricinus ticks and in non-Ixodes ticks from Turkey and Albania was determined by polymerase chain reaction (PCR) and reverse line blot hybridization. In the adult Bulgarian ticks, the prevalence of Borrelia burgdorferi sensu lato infection was approximately 40%, while Borrelia afzelii was the predominant species, representing more than half of all Borrelia-positive ticks. Ehrlichia and Anaplasma species were detected in 35% of the adult Ixodes ricinus ticks and in 10% of the nymphs. Sequence analysis of PCR products reacting with the Anaplasma phagocytophila probe revealed a 16S rRNA gene identical to that of the Anaplasma phagocytophila prototype strain. Ehrlichia and Anaplasma species were found in approximately 7% of the non-Ixodes ticks. Sequence analysis of some of these samples revealed the presence of Anaplasma ovis, Ehrlichia canis, and a species closely resembling Ehrlichia chaffeensis. About half of all adult ticks examined and approximately 20% of all nymphs were infected with Rickettsia species. In Ixodes ricinus ticks, Rickettsia helvetica and a Rickettsia species designated as IRS3 were found in high prevalence. Rickettsia conorii was found in virtually all non-Ixodes tick species from Albania and Turkey. The results of this study show that many tick-borne diseases are most probably endemic in the Balkan area. Furthermore, the results suggest that there is a considerable chance for simultaneous transmission of tick-borne pathogens to human beings.