An ELISA containing a purified flagellar antigen from Borrelia burgdorferi (FLA-ELISA) was evaluated. The FLA-ELISA, detecting IgM and IgG together, did not have adequate specificity by itself. Good accuracy was obtained, however, when the FLA-ELISA was the first step in a two-step protocol that used immunoblotting as a conditional second test. Samples that scored positive or equivocal by the FLA-ELISA were evaluated with separate IgM and IgG immunoblots. The sensitivity of the two-step process for patients with erythema migrans or with later manifestations of Lyme disease was 64% and 100%, respectively. The specificity for health blood donors was 100% and was 90% for the aggregate of all persons with illness that may cause serologic cross-reactivity (98% if the samples from relapsing fever patients were excluded). Test precision was 96% overall, 99% for Lyme disease case serum samples, 100% for specimens from blood donors, and 88% for samples from persons with other illness.