The vls locus of Borrelia burgdorferi B31 consists of 15 silent cassettes and one expression site (vlsE), and the presence of the encoding plasmid lp28-1 correlates with high infectivity. Recombination between the expression cassette and silent cassettes occurs in vivo, and this process may enable B. burgdorferi to evade the immune response. To determine the characteristics of the vls loci in other Borrelia strains, we have cloned and characterized the vls silent cassette loci of Borrelia garinii Ip90 and Borrelia afzelii ACAI, consisting of 11 vls silent cassettes and 14 vls silent cassettes respectively. The silent cassettes share 90-97% nucleotide sequence identity with one another within the Ip90 vls locus and 84-91% within the ACAI vls locus. In both organisms, the silent cassettes resemble the B31 Vls sequences in overall amino acid similarity (50-65%) and in the presence of six variable regions interspersed between six relatively invariant regions. The vlsE expression sites of these two strains have not been isolated, but transcripts of vlsE were detected by reverse transcriptase-polymerase chain reaction for both Ip90 and ACAI. In addition, the occurrence of sequence variation within the vlsE cassette region of these transcripts was verified. This study indicates that the vls loci present in B. garinii Ip90 and B. afzelii ACAI have characteristics similar to those found in B. burgdorferi B31.