Borrelia burgdorferi, the cause of Lyme disease, has two major outer-membrane proteins, OspA and OspB, which act as surface antigens. A 49-kilobase linear plasmid contains the genes that encode for these surface proteins. Direct examination of denatured plasmid molecules has revealed single-stranded circles with a circumference of approximately 100 kilobases (about twice the length of the linear duplex molecule), a finding that indicates the plasmid strands have covalently closed ends. This form of DNA, while present in eukaryotic organisms and their viruses, has not been observed in a prokaryotic organism. Plasmid heterogeneity has been observed in strains with surface proteins of similar molecular weights and similar monoclonal antibody reactivity. Thus, plasmid analysis may prove a sensitive tool for differentiating strains of B. burgdorferi. Furthermore, since loss of plasmids in vitro has been correlated with loss of the ability of many-passaged strains to cause infection, borrelial plasmids may encode for virulence factors as well.