ABSTRACTHydrogen peroxide (H2O2) is a by-product of glycerol metabolism in mycoplasmas and has been shown to cause cytotoxicity for cocultured eukaryotic cells. There appears to be selective pressure for mycoplasmas to retain the genes needed for glycerol metabolism. This has generated interest and speculation as to their function during infection. However, the actual effects of glycerol metabolism and H2O2production on virulencein vivohave never been assessed in anyMycoplasmaspecies. To this end, we determined that the wild-type (WT) Rlowstrain of the avian pathogenMycoplasma gallisepticumis capable of producing H2O2when grown in glycerol and is cytotoxic to eukaryotic cells in culture. Transposon mutants with mutations in the genes present in the glycerol transport and utilization pathway, namely,glpO,glpK, andglpF, were identified. All mutants assessed were incapable of producing H2O2and were not cytotoxic when grown in glycerol. We also determined that vaccine strains ts-11 and 6/85 produce little to no H2O2when grown in glycerol, while the naturally attenuated F strain does produce H2O2. Chickens were infected with one of twoglpOmutants, aglpKmutant, Rlow, or growth medium, and tracheal mucosal thickness and lesion scores were assessed. Interestingly, allglpmutants were reproducibly virulent in the respiratory tracts of the chickens. Thus, there appears to be no link between glycerol metabolism/H2O2production/cytotoxicity and virulence for thisMycoplasmaspecies in its natural host. However, it is possible that glycerol metabolism is required byM. gallisepticumin a niche that we have yet to study.