A 12.9 kb plasmid, pVT2, from a clinicalMycobacterium aviumisolate, MD1, was cloned and radiolabeled for use as a DNA probe to examine the relatedness of plasmids inM. aviumcomplex. That probe hybridized with plasmids isolated fromM. aviumcomplex strains from the environment (7 of 16) and from non-acquired immunodeficiency syndrome (AIDS) (10 of 17) and AIDS (5 of 6) clinical isolates. The similarity of plasmids from the environment with those from patients supports the hypothesis that the environment is a source of humanM. aviumcomplex infection. More striking was the observation that pVT2 hybridized with every plasmid (13 of 13 clinical and 5 of 5 environmental isolates) of 13.5 kb or smaller. A second probe, consisting of a 15.3 kb plasmid (pLR7) from another clinical isolate of theM. aviumcomplex, hybridized with plasmids of 15.3 to 25 kb from environmental and clinical (AIDS and non-AIDS) isolates. There was no hybridization between pVT2 and pLR7. Thus, these two probes define two different groups of small mycobacterial plasmids.