In order to identify antigenic proteins of Mycoplasma gallisepticum, monoclonal antibodies (MAbs) against virulent M. gallisepticum R strain were produced in mice. MAb 35A6 was selected for its abilities to inhibit both growth and metabolism of M. gallisepticum in vitro. The MAb recognized a membrane protein with an apparent molecular mass of 120 kDa. The corresponding gene, designated the mgc3 gene, was cloned from an M. gallisepticum genomic DNA expression library and sequenced. The mgc3 gene is a homologue of the ORF6 gene encoding 130-kDa protein in the P1 operon of M. pneumoniae and is localized downstream of the mgc1 gene, a homologue of the P1 gene. To assess the characteristics of MGC3 protein, all 10 TGA codons in the mgc3 gene, which encode a tryptophan in the Mycoplasma species, were replaced with TGG codons, and recombinant fowlpox viruses (FPV) harboring the altered mgc3 gene were constructed. One of the recombinant FPVs was improved to express MGC3 protein on the cell surface in which the signal peptide of MGC3 protein was replaced with one from Marek's disease virus gB. These results should provide the impetus to develop a vaccine based on MGC3 protein which can induce antibodies with both growth inhibition and metabolic-inhibition activities using a recombinant FPV.