The prevalence of Borrelia burgdorferi sensu lato (s.l.) in 761 Ixodes ricinus ticks collected in different localities of southern Bohemia was investigated. The minimum infection rate of nymphs, males and females as determined by dark-field microscopy was 4.8% in 1997. The total B. burgdorferi prevalence determined in ticks in 1998 was 11.04%. A significant local variability of the prevalence of B. burgdorferi was not found. Nine isolates were identified at a genospecies level by immunoblotting with prepared monoclonal antibodies (Mabs). The most prevalent genospecies in ticks was B. burgdorferi sensu stricto (s.s.) (6 strains). Two tick isolates reacted with Mabs specific to both B. garinii and B. afzelii and one isolate was detected as a mixed culture of B. burgdorferi s.s. and B. garinii. Considerable variability in protein composition among the strains obtained was found to exist. Five B. burgdorferi genospecies-specific Mabs were prepared and used for the identification of spirochaete isolates. Mabs 9B8 and 6C5 reacted with OspA and OspB proteins of B. burgdorferi s.s., while 4D7 and 1D11 recognised OspA protein of B. garinii and 9D2 specifically reacted with the OspB protein of B. afzelii. In addition, Mab 5H4 reacted with flagellin of all the above-mentioned genospecies of B. burgdorferi s.l.