Bacteria of the Mycobacterium avium, M. intracellulare, and M. scrofulaceum group (MAIS) are opportunistic human pathogens and widespread in the environment. The first objective of this study was to demonstrate that plasmids from clinical isolates are closely related to plasmids from environmental MAIS isolates. A 12.9 kb plasmid, pVT2, from a clinical M. avium isolate, MDl, was cloned and used a a DNA probe to examine the relationship of MAIS plasmids. The pVT2 probe hybridized with plasmids isolated from MAIS strains from the environment, from patients without AIDS with pulmonary infections, and from AIDS patients with disseminated MAIS infections. Similar results were seen with a second probe derived from pLR 7, a 15.3 kb plasmid from clinical M. intracellulare strain LR 113. The similarity of plasmids from environmental and clinical isolates supports the hypothesis that the environment is a source of MAIS infection in humans.