Many lipoproteins are expressed on the surfaces of mycoplasmas, and some have been implicated as playing roles in pathogenesis. Family 2 lipoproteins of Mycoplasma pneumoniae have a conserved "mycoplasma lipoprotein X" central domain and a "mycoplasma lipoprotein 10" C-terminal domain and are differentially expressed in response to environmental conditions. Homologues of family 2 lipoproteins are Mycoplasma specific and include the lipoprotein of Mycoplasma gallisepticum, encoded by the MGA0674 gene. Comparative transcriptomic analysis of the M. gallisepticum live attenuated vaccine strain F and the virulent strain R(low), reported in this study, indicated that MGA0674 is one of several differentially expressed genes. The MGA0674-encoded lipoprotein is a proteolytically processed, immunogenic, TX-114 detergent-phase protein which appears to have antigenic divergence between field strains R(low) and S6. We examined the virulence of an R(low) Delta MGA0674 mutant (P1H9) in vivo and observed reduced recovery and attenuated virulence in the tracheas of experimentally infected chickens. The virulence of two additional R(low) Delta MGA0674 mutants, 2162 and 2204, was assessed in a second in vivo virulence experiment. These mutants exhibited partial to complete attenuation in vivo, but recovery was observed more frequently. Since only Mycoplasma species harbor homologues of MGA0674, the gene product has been renamed "Mycoplasma-specific lipoprotein A" (MslA). Collectively, these data indicate that MslA is an immunogenic lipoprotein exhibiting reduced expression in an attenuated strain and plays a role in M. gallisepticum virulence.