The recent isolation and characterization of two plasmids from Mycoplasma mycoides subspecies mycoides has opened up new possibilities for studying mycoplasmal genetics. In order to facilitate the development of a genetic system in M. mycoides subsp. mycoides, parameters of polyethylene glycol (PEG)-mediated transformation were examined, as existing protocols prove very inefficient in this organism. The effects of PEG concentration, DNA concentration, presence of Ca2+ ions, and choice of buffers on the transformation of the Tn916-containing plasmid pAM120 into M. mycoides subsp. mycoides were examined. The stability of Tn916 in the M. mycoides subsp. mycoides chromosome was also evaluated. The optimal PEG concentration (53-62% ( w v)) in the transformation mixture was substantially higher than the PEG concentration reported to be optimal for transformation of other mycoplasmas (36% ( w v)). The PEG concentrations used here were also higher than the concentration used to promote transformation or fusion of gram-positive bacterial protoplasts. A necessity for the presence of Ca2+ ions for optimal transformation was shown, as was the possible involvement of cell culture growth stage. Our results demonstrate the need for expanding current transformation techniques for mycoplasmas. Studies also indicate that once Tn916 inserts into the M. mycoides subsp. mycoides chromosome, it can transpose to other sites at a relatively high frequency. © 1991.