It is difficult to distinguish pulmonary disease due to Mycobacterium avium complex (MAC) from that due to other mycobacteria, such as Mycobacterium tuberculosis and Mycobacterium kansasii. We developed an enzyme immunoassay (EIA) for diagnosis of MAC pulmonary diseases that uses glycopeptidolipid (GPL) antigens specific for MAC, and we used it for diagnosis in immunocompetent patients. The mean optical densities (+/- standard deviation) of serum immunoglobulin G antibodies to GPLs in patients with MAC disease, MAC colonization, M. kansasii disease, and tuberculosis and in healthy subjects were 0.778+/-0.784, 0.042+/-0.035, 0.059+/-0.035, 0.071+/-0.035, and 0.030+/-0.027, respectively. A significant increase in the level of anti-GPL antibodies was detected in patients with MAC disease. The level of anti-GPL antibodies reflected disease activity, because the level was decreased in culture-negative patients who had conversion of culture results. When a cutoff level of seropositivity (0.119) was defined, the sensitivity of EIA for diagnosis of MAC disease was 92.3%, and the specificity was 96.7%. Measurement of serum anti-GPL antibodies is useful for both the diagnosis of and assessment of activity in MAC disease.