In view of the significant sequelae associated with Lyme borreliosis, there is a need for timely and accurate diagnosis of erythema migrans (EM). Although Borrelia burgdorferi can be cultured from biopsies of EM lesions, immunodiagnostic testing is more widely available. Four immunoserologic methods were studied by using the sera of 51 patients with EM lesions that were culture positive for B. burgdorferi. Nineteen patients had single primary lesions, and thirty-two had multiple secondary lesions. At the time of biopsy, 40 patients, 8 with primary lesions and all patients with secondary lesions, were seropositive by an immunoglobulin M (IgM) indirect fluorescent-antibody (IgM IFA) test (Bion Enterprises). Twenty-three patients were seropositive by a whole-cell fluorescence enzyme immunoassay (EIA) (BioWhittaker, Inc.), twenty-two were positive by immunoblotting (ViroStat, Inc.), and one was positive by a P39 recombinant EIA (P39 EIA) (General Biometrics, Inc.). Sera from various patient control groups were tested: rheumatoid arthritis (n = 19), infectious mononucleosis (n = 20), systemic lupus (n = 22), syphilis (n = 13), streptococcal sequelae (n = 20), and healthy subjects (n = 16). None of these sera reacted with the IgM IFA test or P39 EIA. Fifteen reacted with the fluorescence EIA. We conclude that the IgM IFA test is an effective and reliable assay for the diagnosis of EM, particularly for patients with secondary lesions. Immunoblot, fluorescence EIA, and P39 EIA lack the sensitivity to reliably diagnose EM.