Mycoplasma arthritidis is a rat pathogen causing a severe polyarthritis. The study of its pathogenic mechanisms has been hampered by the lack of genetic systems for use with M. arthritidis. Described here are procedures for genetic transformation of M. arthritidis and conjugal transfer of Tn916 from an enterococcal donor to M. arthritidis. The location of Tn916 insertion sites in the mycoplasmal chromosome was random, suggesting that Tn916 may be useful as an insertional mutagen in this organism. Additionally, a restriction and modification system was identified which presented a strong barrier to gene transfer. For transformation, the restriction system was circumvented by using DNA that was modified in vitro with the appropriate site-specific methylase (AluI).