The genomic DNA patterns (genotypes) of 55 episodes of late positive sputum isolates, collected after >or=4 consecutive months of negative sputum cultures, in prospective macrolide treatment trials of Mycobacterium avium complex (MAC) lung disease were assessed by pulsed-field gel electrophoresis (PFGE). Having >or=2 cultures positive for MAC after completion of therapy was documented 23 times; of 20 episodes studied by PFGE, 17 (85%) represented new genotypes (i.e., new infections), and 87% occurred in patients with nodular bronchiectasis. With >or=2 positive cultures after therapy was stopped prematurely, 6 (86%) of 7 episodes were relapses. Single positive cultures after completion of therapy occurred 16 times; only 1 (6%) was predictive of a subsequent relapse. No late isolates were macrolide resistant. Thus, relapses of MAC lung disease with these macrolide regimens are unusual, and most infections after completing therapy resulted from new strains in patients with nodular bronchiectasis.