Mycoplasma ovipneumoniae is a globally distributed pathogen associated with pneumonia in domestic and wild caprids. It is the primary cause of chronic non-progressive pneumonia (CNP) in domestic sheep in New Zealand. CNP is associated with decrease in growth rates as well as additional feed requirements to increase the live weight of lambs for market. Further economic losses from CNP is from downgrading carcasses due to pleurisy. The absence of overt clinical symptoms associated with CNP and resistance of M. ovipneumoniae to many antibiotics, which is due to lack of a cell wall, hinders detection and treatment of the disease. Isolates of M. ovipneumoniae have been obtained from New Zealand farms with a history of ovine pneumonia. These isolates are being used to identify immunogenic cell surface proteins and determine their potential use as diagnostic antigens and/or sub-unit vaccine proteins. Membrane fractions were prepared from three of the NZ isolates of M. ovipneumoniae and immunoblotted with antisera produced in sheep against the mycoplasmas. Eight immunogenic proteins were identified, including EF-Tu and HSP70, mycoplasma proteins known to induce immunological responses in other animal species however EF-Tu had not previously been described as immunogenic in sheep. Six of the proteins have not been reported previously. EF-Tu and four of the novel proteins were produced as recombinant proteins. Sheep were vaccinated with the proteins, whole cells or membrane fractions prepared from M. ovipneumoniae and antisera was obtained for measurement of antibody responses and antigen-specific IFN-g production in antigen-stimulated whole blood cultures. Immunofluorescence microscopy with the antisera confirmed localisation of the proteins on the surface of the mycoplasmas.