An immunogenic region of the Borrelia burgdorferi flagellin encompassing amino acids 197-273 and designated 41-G was evaluated as an antigen in an enzyme immunoassay (EIA) for Lyme disease on a routine basis in a reference laboratory. Sera that tested positive for Lyme disease by EIA using 41-G or the whole-cell Borrelia burgdorferi lysate as the antigen were also evaluated by immunoblot for reactivity with Borrelia burgdorferi, and the patient's clinical history was determined retrospectively by a questionnaire distributed to the referring physician. The sensitivity of the 41-G based EIA for the serologic diagnosis of Lyme disease, when compared with that of the Borrelia burgdorferi lysate EIA, was 70% (35 of 50). These data demonstrate that 41-G has utility as an antigen in EIA, although the sensitivity is at present less than that of the assay employing the Borrelia burgdorferi whole-cell lysate.