Linear plasmids are widely distributed in isolates of Borrelia burgdorferi, but the prevalence of supercoiled circular (SC) plasmids has not been clearly established. Enriching for SC plasmids on ethidium bromide density-gradients revealed that a low passage of strain Sh-2-82 of B. burgdorferi has at least six SC plasmids (8.4, 8.8, 20, 26, 29 and 51 kb). In addition, several of the SC plasmids were observed by electron microscopy to form concatenated structures. Because of the high incidence of coupled SC plasmids and the effect of such structures on plasmid mobility, we suggest that they could be mistaken for a single DNA species having a high molecular weight when total DNA from B. burgdorferi is examined by agarose-gel electrophoresis. Of the six SC plasmids in strain Sh-2-82, four could still be detected after two years of continuous in vitro passaging in BSK II medium. The two SC plasmids lost after a maximum of 20 passages, pBBC1 (8.4 kb) and pBBC2 (8.8 kb), shared a high degree of DNA sequence similarity, suggesting that their apparent instability may be a unique feature of these closely related plasmids. Two higher passaged variants of strain Sh-2-82, P20 and P202, were shown by hybridization to lack pBBC1 and pBBC2 sequences and were unable to infect the white-footed mouse, Peromyscus leucopus. However, six of 14 unrelated isolates that infected this rodent and Syrian hamsters also lacked pBBC1 and pBBC2 sequences. None of five unrelated but highly passaged non-infectious isolates carried either pBBC1 or pBBC2 sequences. These data indicate that pBBC1 and pBBC2 do not encode constitutively expressed proteins required for infectivity in our experimental system. Nevertheless, eight of the 13 infectious strains examined did carry pBBC1 and/or pBBC2, indicating that these two closely related plasmids can be detected in a wide range of unrelated isolates of B. burgdorferi.