During the years 1995-1996, a total of 1,743 overwintering Culex pipiens biotype molestus female mosquitoes were tested for the presence of spirochetes in several localities in South Moravia, Czech Republic.The spirochetes were observed in 5% of the mosquitoes investigated. One of the five isolated strains of spirochetes (BR-84) was identified as Borrelia afzelii. The potential role of mosquitoes in the ecology and epidemiology of Lyme disease (LD) borreliae should be further investigated.