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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.