Transposons Tn4001 and Tn916 have been introduced into M. pulmonis. Additionally, the antibiotic resistance genes of both transposable elements have been cloned and integrative plasmids constructed that carry chromosomal DNA as a region of homology. Transposons and integrative plasmids were used to study polyethylene glycol mediated transformation in M. pulmonis and the optimal transformation frequencies were obtained in a mixture containing 35.5% PEG, 10 ug plasmid DNA, 10 ug yeast tRNA and 1 x 10[superscript]8 cells. Transformation frequencies were enhanced four- to five-fold by preincubating cells at 0° C for 30 min. in CaCl[subscript]2 prior to transformation. Under these conditions Tn916 and Tn4001 could be introduced at 1 x 10[superscript]-6 and 5 x 10[superscript]-5 respectively and integrative plasmids could be introduced at frequencies between 1 x 10[superscript]-4 and 1 x 10[superscript]-6. Integrated plasmids could be retrieved in Escherichia coli and are the first shuttle vectors and cloned genes introduced in the Mollicutes. A system for recombinant DNA manipulations in M. pulmonis is described that targets previously integrated plasmids for homologous recombination with a cloning vector carrying foreign DNA. Recombinant molecules introduced in this way were found to be stably integrated and maintained;A genetic exchange system is described for M. pulmonis that occurs both in liquid culture and on agar surfaces. Genetic exchange was limited to selected strains of M. pulmonis and did not occur in M. gallisepticum or Acholeplasma laidlawii indicating that genetic exchange was not a general feature of mycoplasmas. In M. pulmonis transposon and integrated plasmid markers were exchanged at frequencies between 1 x 10[superscript]-4 and 1 x 10[superscript]-8. Southern hybridization of transconjugants indicated that the markers resided in identical locations as in the parents and resulted from homologous recombination. Genetic exchange was not effected by the addition of DNase, polyethylene glycol, EDTA or CaCl[subscript]2 but was effected by the addition of trypsin. These data support a conjugation like mechanism involving trypsin sensitive components for genetic exchange in M. pulmonis.