Acute phase serum samples from 23 patients with Mycoplasma pneumoniae pneumonia were examined for specific antibodies with microparticle agglutination (MAG)-assay, complement fixation (CF)-test, IgM-specific ELISA, and Immunocard-based IgM-EIA. A novel 3-h IgA-ELISA (SeroMP-IgA) was tested to assess its diagnostic value. For MAG, CF, IgM-ELISA, card-based IgM-EIA, and IgA-ELISA positive results were obtained in 87%, 87%, 91%, 87%, and 100%, respectively. Overall concordance was 78%. Specificity testing of 46 healthy blood donors revealed an optimal diagnostic cut-off range of 22-30 IgA-ELISA binding units (BU)/mL resulting in 91%-100% specificity and 100%-96% sensitivity for serologic diagnosis of acute M. pneumoniae infection.