Mycoplasma is in the class of Mollicutes which are characterized by their lack of cell wall and have evolved drastically by reduction of the genome size. Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia and is important cause of economic losses in swine industry. While Mycoplasma bovis, a pathogen of ruminants, causes a variety of disease usually from mastitis, respiratory disease, arthritis and urogenital tract infections. Regardless of years of research, the design of a vaccine to protect both swine and cattle has not been effective. Real progress in the study of molecular mechanisms of pathogenicity also require efficient molecular tools to look at gene function. Among the surface proteins of M. hyopneumoniae there are a few involved in virulence that have been extensively studied, P97 and P102. These proteins are directly related to the attachment of the organism to the swine respiratory tract. In this study we looked at recombination as a method of genetic manipulation to construct null mutants of P97. These mutants were characterized in terms of loss of P97/P102 proteins by immunoblot using monoclonal antibodies, ability to bind purified swine cilia and adherence to swine PK15 cells. We also looked at the development of a β-galactosidase strain in M. bovis and M. hyopneumoniae to target for recombination studies assessing recombination efficiency. Various forms of transforming DNA were tested for generating knockout mutants of P97 or β-galactosidase through recombination. This included intact plasmid DNA, single-stranded DNA and linear DNA with and without a heterologous recA gene. Our results indicate that recombination can be used to generate site-specific mutants in M. hyopneumoniae. P97/P102 mutants are deficient in cilia binding and PK15 cell adherence and lack the characteristic banding pattern seen in immunoblots developed with the anti-P97 monoclonal antibody. We were also able to develop a genetic system in M. bovis to create a non-specific mutation into a variety of genes by the insertion of a transposon carrying the lacZ gene. This approach opens the way to study pathogen-host interactions and virulence factors of M. bovis and M. hyopneumoniae and develop new genetically defined vaccine strains.