Low (7th) and high (298th/304th) in vitro passages (cultivated over a period of 3 years) of two human Borrelia burgdorferi sensu lato strains, PKo (B. afzelii) and PBi (B. garinii) were compared by pulse-field gel electrophoresis, Southern blot, sequencing of the ospA gene, SDS-PAGE and Western blot. Digestion of genomic DNA with ApaI, BssHII, KspI, MluI, SmaI and XhoI did not reveal any differences between low and high passages. The loss of two linear plasmids with sizes of 6 and 31 kbp was detected in strain PKo between passages 34-50 and 101-304, respectively, whereas the ospA-carrying plasmid remained unchanged. In contrast, analysis of linear plasmid profiles obtained from low and high passages of B. garinii strain PBi showed no differences. Sequence analysis of the ospA gene demonstrated no difference in the strain PBi and one nucleotide exchange in the strain PKo when low and high passages were compared. The observed transition (G-A) in the third codon position did not alter the amino acid sequence. However, the rate of expression of the outer surface proteins OspA, OspB and OspC of strain PKo during low and high stages of cultivation varied significantly. In summary, our data suggest that the B. burgdorferi sensu lato genome is stable during long-term in vitro cultivation.