A procedure for transformation of the murine pathogen Mycoplasma pulmonis with plasmid pAM120 was developed. This plasmid replicates in Escherichia coli and contains the gram-positive transposon Tn916. The transformation protocol also proved effective for the swine pathogen Mycoplasma hyorhinis. The tetracycline resistance determinant of Tn916 was expressed in transformed mycoplasma cells, and Tn916 was found inserted into numerous sites in the recipient chromosomes of M. pulmonis and M. hyorhinis, indicating that transposition had occurred. Interestingly, some transformants of M. pulmonis and M. hyorhinis contained cointegrate structures which apparently had a complete copy of the entire donor plasmid (pAM120) inserted into the recipient chromosome. Subsequent transposition of inserted Tn916 was observed in passaged clones of transformed M. pulmonis. © 1988.