A Borrelia burgdorferi chromosomal gene encodes a 30-kDa antigen (P30) that has considerable homology with periplasmic substrate-binding proteins of Gram-negative bacteria, and is recognized by antibodies in sera from a subset of patients with Lyme disease and from B. burgdorferi-infected mice. The p30 gene is 801 nucleotides in length and P30 contains 267 amino acids, with a predicted molecular mass of 30 kDa. The P30 amino acid region 36-258 has homology to conserved domains of the oligopeptide permease A of Gram-negative bacteria. Immunofluorescence studies using murine anti-P30 serum suggest that P30 is on the outer surface of B. burgdorferi. P30 expression could be detected in representatives of all 3 subspecies of B. burgdorferi sensu lato, but not in all of the tested strains. Antibodies to P30 were detected in sera of 18 out of 82 patients (22%) with Lyme disease, including individuals with early- or late-stage infection. Although antibodies to P30 are present in the sera of C3H/HeN mice infected with B. burgdorferi for at least 90 days, immunization with recombinant P30 does not protect mice from infection. We conclude that P30 is a putative substrate-binding protein of B. burgdorferi and is immunologically recognized in human and murine Lyme borreliosis.