Mycoplasma gallisepticum causes respiratory disease in avian species, but little is known about its mechanism(s) of pathogenesis. These studies were undertaken in order to develop genetic systems for analysis of potential virulence factors. M. gallisepticum was transformed with plasmids containing one of the gram-positive transposons Tn916 or Tn4001, which inserted randomly into the mycoplasmal chromosome. Plasmids containing cloned chromosomal DNA were also constructed and tested for integration into regions of DNA homology derived either from chromosomal fragments or from the gentamicin resistance marker from Tn4001. These studies demonstrate that M. gallisepticum is amenable to transformation with both transposons and integrative vectors.