Mycoplasma fermentans is currently being examined as an agent potentially associated with human disease. Several strains of M. fermentans were isolated from patients with respiratory tract disease and AIDS. Two of these clinical strains, M64 and SK6, were triple-filter-cloned and designated as the parental clones in this study. Genomic DNA of randomly picked subclones in four and five subsequent generations passed from the parental M64 and SK6 clones were analyzed by using a radiolabeled M. fermentans-specific insertion sequence (IS)-like element as the probe. The hybridization patterns of DNA restriction fragments revealed high frequencies of chromosomal changes accompanied with excision or new insertion of the IS-like element in M. fermentans chromosome. The findings indicate M. fermentans has an effective mechanism(s) to produce a rapid gene rearrangement that may be mediated by one or more copies of the IS-like element.