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A total of 491 Ixodes vespertilionis and 8 Ixodes ricinus collected from bats and cave walls in southern Poland between 2010 and 2012 were examined by the polymerase chain reaction for tick-transmitted pathogens. PCR analysis for Borrelia burgdorferi s.l., Rickettsia spp., and Anaplasma phagocytophilum yielded negative results for all I. vespertilionis. DNA of Rickettsia helvetica was detected in three specimens of I. ricinus attached to Rhinolophus hipposideros or Myotis myotis, while Borrelia garinii was found in one tick parasitizing Myotis daubentonii. These pathogens were recorded for the first time in hard ticks that parasitized bats.