A total of 662 samples (winter period: 469; summer period: 193 specimens) of female mosquitoes of the genus Culex, Aedes and Anopheles were collected during the period March 2000-April 2001 from the locality of Vysoké Mýto (Eastern Bohemia, Czech Republic). They were examined by dark field microscopy for the presence of spirochetes. The motile spirochetes were observed in 4.2% of all species of investigated mosquitoes. One spirochetal strain out of the 8 isolation attempts (BRZ14) was obtained (cultivation rate was 12.5%) and the spirochetal strain was then successfully cultivated and identified using PCR for the presence of Borrelia burgdorferi s.l., and subsequently with the RFLP as genomospecies Borrelia afzelii. This strain was derived from overwintering Culex (Culex) pipiens biotype molestus female mosquitoe. This is apparently one of the sporadic cases of the occurrence of pathogenic borreliae in haematophagous arthropods, other than Ixodes ricinus complex ticks.