The presence of a membrane lipoprotein homologous to the P48 of Mycoplasma agalactiae was investigated in different Mycoplasma bovis isolates selected by geographical locations and biological properties. Its potential as a diagnostic tool was also discussed. The presence of a specific signal observed in all M. bovis field isolates probed with a rabbit antiserum raised against the M. agalactiae recombinant P48 demonstrated that this protein is structurally and antigenically conserved within the M. bovis cluster. No signal was detected when testing six different mycoplasma species found in cattle. The p48 gene was identified by PCR approach and partially sequenced. Full length gene sequence was obtained by direct bacterial chromosome sequencing. Five UGAs were selectively mutated into UGG and the full length mutated gene, lacking the signal peptide, was cloned and expressed in Escherichia coli. The purified recombinant antigen (r-P48) was evaluated as a potential marker of infection using a panel of 86 well-characterized sera from experimentally and naturally infected cattle. Specific IgM antibodies were detected within 6-9 days after experimental infection followed by an IgG response lasting from the third/fourth week after contact. Although antibody titers were well below those observed in sheep or goats infected with M. agalactiae, results suggest that M. bovis r-P48 can be used as a specific marker of infection.