Mycoplasma hyopneumoniae is the primary aetiological agent of enzootic pneumonia, a chronic respiratory disease of considerable economic importance to pig industries around the world. Advances in our understanding of the pathogenesis of this bacterium at the molecular level have been hampered by a lack of genetic tools to allow the manipulation of the mycoplasma genome. We have recently reported the first successful transformation of M.hyopneumoniae with self-replicating plasmids containing the predicted M.hyopneumoniae origin of replication (oriC) and the tetM determinant, conferring tetracycline resistance to transformed bacteria. We have used our oriC plasmids to optimise the conditions required for transformation of the 232 strain of M.hyopneumoniae by electroporation and the successful culture of transformants in solid and liquid medium. Using our optimised conditions, we have determined the susceptibility of further isolates of M.hyopneumoniae to transformation. Self-replicating plasmids have been successfully used to generate targeted gene mutants in other mycoplasmas by homologous recombination. With this in mind, we have determined the stability of our oriC containing plasmids over serial passages in liquid culture. In an attempt to reduce the integration of plasmid DNA into the mycoplasma genome, we have determined the minimal size of the oriC region that allows plasmid maintenance as extrachromosomal DNA. As an alternative strategy to reduce integration of plasmid DNA by homologous recombination, we have produced plasmids containing the oriC regions of phylogenetically related mycoplasma species and determined their ability to transform M.hyopneumoniae. It is anticipated that our plasmids will be useful tools in allowing the generation of targeted gene mutants in M.hyopneumoniae. Furthermore, in demonstrating the amenability of M.hyopneumoniae to transformation and in optimising the conditions necessary to do so, our work is an important step in the development of other strategies such as transposon mutagenesis, that will greatly assist our study of this pathogen.