DNA fragments containing the VMP-like sequence (Vls) were cloned from Borrelia burgdorferi strain 297. Analyses by PCR, PFGE, and Southern hybridization revealed that the Vls sequences existed in multi-copies on the 20-kb borrelial plasmid, but not on chromosomes or other plasmids. One Vls unit of the strain 297 was about 669 bases, and predicted peptides length was 223 amino acids. Homologues of the Vls fragment were detected in three B. burgdorferi strains, a B. garinii strain 20047, and a B. afzelii strain P/Gau. A recombinant VlsII protein prepared in Escherichia coli strain JM109 reacted with antibodies that existed in three of five patients, by immunoblotting. These results suggested that the Vls of B. burgdorferi is expressed in Lyme disease patients.