Sera from patients with disease caused by the Mycobacterium avium complex (M. avium and M. intracellulare), M. kansasii, or M. tuberculosis and from subjects who did not have a mycobacterial disease were tested by enzyme-linked immunosorbent assay against peptidoglycolipid antigens representing each of the 15 most common serovars of the M. avium complex and against crude protein antigen extracts of M. avium and M. tuberculosis. The highly specific peptidoglycolipid antigens yielded positive reactions in 83% of M. avium complex patients, 57% of active-tuberculosis patients, and 14% of subjects without mycobacterial disease. Reactions to more than 1 of the 15 peptidoglycolipid antigens were found only in patients with infections caused by mycobacteria, suggesting that a mycobacterial pulmonary lesion is readily colonized by mycobacteria other than the one that initiated the lesion. The two crude mycobacterial protein antigens were highly cross-reactive, with little if any capacity to discriminate between infections caused by any of the mycobacteria studied. Moreover, they did not appear to be more sensitive than the peptidoglycolipids. The data suggest that it is unlikely that a practical and reliable serological test can be developed that will distinguish between transient subclinical infection and significant disease caused by common environmental mycobacteria, such as members of the M. avium complex. Success in developing such a test for nonenvironmental mycobacteria, such as M. tuberculosis, appears more likely.