The recombinant clone expressing a 60 kDa (P60) antigen was isolated from Escherichia coli by screening a lambda EMBL3 genomic library using rabbit produced antiserum against Mycoplasma hyopneumoniae. Sequence analysis revealed that an interrupted (by a UGA codon) open reading frame coding for a 72 kDa protein (P72) may contain the P60 antigen gene. Western blot analysis with an anti-P60 monospecific antibody confirmed the presence of a P72 antigen from the total protein of M. hyopneumoniae, and a 72 kDa protein was also expressed in E. coli after changing the codon (UGA to UGG) by site-directed mutagenesis. BLAST (Basic Local Alignment Search Tool) comparison showed that the amino acid sequences of P72 share approximately 70% homology with the phosphotransferase enzyme I (PTSI) of bacteria and other mycoplasma species. The biological function of the P72 cytosolic protein was further confirmed by complementation using an E. coli ptsI mutant. The bacterial phosphoenolpyruvate-sugar phosphotransferase system (PTS) is known to mediate the uptake and phosphorylation of carbohydrates and to be involved in signal transduction. The immune responses of specific pathogen free (SPF) pigs and farm animals toward this unique antigen were observed. The transcription start positions of the PTSI gene were determined in M. hyopneumoniae and E. coli by primer extension experiments and the promoter site was also predicted.