Different media have been utilized for borrelial cultivation. The aim of the present study was to evaluate the isolation rate of Borrelia burgdorferi sensu lato from two commonly used media, i.e. modified Kelly-Pettenkofer (MKP) and Barbour-Stoenner-Kelly II (BSK-II) medium, and to compare the isolated strains with regard to their phenotypic and genotypic characteristics. Skin biopsy specimens of 2 x 2 x 4 mm were taken from the peripheral site of human solitary erythema lesions and were divided in two pieces, one of which was inoculated into MKP and the other one into BSK-II medium. Species analysis of the obtained strains was performed and their plasmid and protein profiles were determined. Borrelia strains were isolated from 48/96 patients (50%) with erythema migrans. We obtained in 26/48 patients (54%) from MKP as well as from BSK-II, in 11 patients (23%) only from MKP, and in another 11 (23%) only from BSK-II medium a positive result. B. afzelii was isolated from 43 patients (23 were positive in both media, nine in MKP only and 11 in BSK-II medium only), while B. garinii was isolated from five patients (in three from both media, in two from MKP only). All strains of the obtained strain pairs were identical according to species and the type within the species. Plasmid profiles were identical in 17/21 B. afzelii strain pairs (81%) and in 1/3 B. garinii strain pairs; in 6/24 strain pairs, distinctions in the number of plasmids or in their molecular mass were present. Differences in the protein profile were found in 7/24 strain pairs (29%). The distinctions were uniform and were limited to the expression of OspC. In conclusion, our study showed comparable Borrelia isolation rates from MKP and BSK-II medium. The results of the present study indicate that human patients with Lyme borreliosis may simultaneously harbor heterogeneous B. burgdorferi s.l. strains.