Technical Abstract: Mycoplasma gallisepticum (MG) is the causative agent of chronic respiratory disease in layer chickens. There are currently three MG vaccine strains approved and commercially available for use in layer chickens, and they include F, ts-11 and 6/85. The F-strain was the first developed and today is the most economic and commonly used vaccine strain in the United States. Mycoplasma gallisepticum vaccine strains ts-11 and 6/85 are safer than the F-strain vaccine and they have little or no potential of spreading from bird to bird. However, ts-11 and 6/85 appear to be less efficacious than F-strain. This may result from the ability of the F-strain to persist for the life of the vaccinated animal. In the present study, mass spectrometry was used to evaluate the proteome of the F-strain. A total of 619 proteins were recognized following stringent statistical criteria for protein identification. Clusters of Orthologous Groups of proteins (COGs) were used to evaluate the function of the identified proteins. Based on COGs analysis 6, 13, and 18% of identified proteins were predicted to be involved in such cellular processes as signaling, metabolism and information processing. However, 61% of proteins identified were not in COGs and 2% were poorly characterized. Preliminary results suggest that proteomics may aid in the identification and characterization of F-strain proteins important in host colonization.