Antibodies against a 35-kDa antigen of Borrelia burgdorferi are detectable in the serum of about half of patients with early Lyme disease. The gene encoding this antigen was isolated from a genomic library of B. burgdorferi B31 (low passage), and full-length expression of the recombinant gene product was achieved in Escherichia coli. Antiserum raised against the recombinant protein was reactive with a B. burgdorferi protein of the same molecular size as the diagnostic 35-kDa antigen cited in an earlier study of criteria for the sero-diagnosis of early Lyme disease. Also, the recombinant protein was reactive with serum from patients with early Lyme disease who were seropositive for the 35-kDa antigen. DNA sequence analysis of the gene indicated an open reading frame of 909 bp encoding a protein with a calculated molecular mass of 34.3 kDa. This gene did not possess the usual initiation codon ATG but rather probably used a TTG codon. The deduced amino acid sequence of the N terminus exhibited a motif similar to that for signal peptides of lipoproteins. Southern blotting revealed a chromosomal location for this gene; and it was specific for B. burgdorferi, B. afzellii, and B. garinii but not for B. hermsii, B. coriaciae, or B. turicatae.