After first finding Borrelia in the midgut of imago mosquitoes, we concentrated on the presence of Borrelia in mosquito development stages-larvae of the third or fourth instar. In the summer season in the years of 2000-2001 a total of 439 Culex (Culex) pipiens pipiens larvae were collected from a barrel of rainwater in the Obrany holiday area of Brno city (East Moravia, Czech Republic). The larvae midgut was observed under dark-field microscopy. Ten DFM positive samples (2.28%) were further analysed using the single-tube nested PCR method for the presence of flagellum DNA sequence specific for Borrelia burgdorferi sensu lato, of which 5 were positive. Borrelian positivity of Culex (C.) pipiens pipiens larvae was 1.14%. One spirochete isolated strain in BSK-H medium was obtained. PCR detection for borrelian DNA of the isolated strain was negative. From these results we can conclude that a low percentage Borreliae can be also found in mosquito larvae and are likely to survive into imago stage.