Sera from 14 groups of chickens inoculated with different laboratory and field strains of Mycoplasma gallisepticum (MG) were used to compare the diagnostic potential of the hemagglutination-inhibition (HI) test and a recently developed monoclonal blocking enzyme-linked immunosorbent assay (ELISA). HI was performed with strain A5969, commonly used as hemagglutinating antigen, and it could detect 62.7% of the inoculated chickens as positive. Of all sera, 83% proved to be positive when examined with the blocking ELISA. The difference between the sensitivities of the two methods was due to group-specific insensitivity of the HI test. None of the sera from groups inoculated with strains K 1501, K 1503, K 503, or K 703 and only half of the sera hem groups inoculated with K 1453 or 236C could inhibit the activity of the A5969 hemagglutinating antigen, indicating antigenic differences between these challenge strains and the diagnostic strain. ELISA detected MG-specific antibodies in every group of sera, although inoculation with variant strains K 503 or K 703 resulted in lower level of antibody production than inoculation with other strains. The monoclonal blocking ELISA can be useful in the serological diagnosis of MG infections, because it is based on a consistently expressed, specific region of MG.