Mycobacterium avium is an intracellular pathogen that is associated with disseminated infection in acquired immunodeficiency syndrome (AIDS) patients. Human monocyte-derived macrophages were infected with M. avium strain 101 and a quinolone (Bay y 3118) was used at 8 micrograms ml-1, a concentration that kills growing bacteria but fails to eliminate static organisms. Infected monolayers were treated with Bay y 3118 for 4 days and viable bacteria obtained from the lysis of macrophages were used to infect other macrophages without passage in media. The procedure was repeated five times, after which seven different subpopulations that failed to grow within macrophages were identified. While the DNA fingerprinting confirmed that all came from the same strain, three protein profiles were observed. Static subpopulations were not killed by cytokine-stimulated macrophages, in contrast to the replicating subpopulation. Three of the static subpopulation strains were shown to be auxotrophic for glutamic acid or methionine. All seven non-duplicating subpopulation strains grew well in complete 7H10 agar. The importance of a static subpopulation of M. avium within macrophages is presently unknown. It is possible, however, that the non-growing bacteria would persist within macrophages.