BACKGROUND : Brucellosis is the most common zoonotic diseases worldwide. The aim of this study was to develop and evaluate the diagnostic performance of an indirect-ELISA (I-ELISA) method based on whole cell Brucella abortus S99 lysates for detection of IgM anti-Brucella antibodies in a human serum. METHODS : The study was conducted in two species-rich endemic areas of Iran (Tehran and Lorestan provinces). Serum samples of 102 patients and 150 healthy individuals were tested by the new kit and the commercial Vircell kit for the presence of anti-Brucella IgM antibodies. The disease status was confirmed by Wright agglutination test. The difference in the mean optical densities (OD) recorded by the new and the Vircell kits for patients and healthy individuals were tested using Two-tailed Student t-test. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the new kit were informed using Receiver operating curve analysis. The results were used to guide the choice of cutoff. Agreements in ODs recorded by the new and the Vircell kit was visually inspected using Bland-Altman plot. RESULTS : The new I-ELISA showed excellent diagnostic performance (sensitivity and PPV = 95.7%, specificity and NPV = 97.8%) for the diagnosis of brucellosis. The cut-off area for the antibody index (AI) was determined to be 8-10, where AIs less than 8 and greater than 10 were considered Brucella-negative and -positive, respectively. AIs of 8-10 show equivocal results, requiring re-testing. The Vircell kit showed low (36.8%) sensitivity and perfect (100%) specificity on the same samples. The Bland-Altman plot showed low agreement between both tests in recording the OD values for the same individuals. CONCLUSIONS : The new I-ELISA based on whole cell Brucella abortus S99 showed a good performance for the detection of Brucella spp. Lack of agreement between the new and the Vircell kit suggest that the performance of ELISA kits might be dependent on the geographical area under study. Hence, validation of the new and the Vircell kits is recommended prior to their implementation in other geographical areas.