Recombinant (r) outer surface proteins (Osp) A and B, flagellin, and an immunogenic region of flagellin (41-G) from Borrelia burgdorferi were evaluated using immunoblot and ELISA for usefulness as substrates in diagnostic testing for Lyme disease. Antibodies to rOspA, rOspB, and r-flagellin were detected by immunoblot and ELISA using the recombinant proteins. Patients with late disease responded to rOspA, rOspB, and r-flagellin, or only to r-flagellin, whereas patients with early disease showed no response or responded only to r-flagellin. Patients who developed antibodies to r-flagellin also responded to 41-G. The data suggest that recombinant B. burgdorferi antigens can serve as substrates for immunoblot and ELISA, which may be helpful in diagnosing Lyme disease.