Introduction: Bovine tuberculosis, a disease caused by infection with Mycobacterium bovis, remains a serious economic problem in several countries and a zoonotic risk in others, partly due to inadequate diagnostic tests. In the British Isles, skin-testing, which measures an animal's response to intradermal injections of tuberculin, is used as an indicator of infection. However, the PPD used is a poorly characterised mixture of mycobacterial antigens, with components which are not species-specific. It is envisaged that the identification of epitopes which are specific to the target organism may advance the development of more specific and sensitive diagnostic tests. In recent years, the use of synthetic overlapping peptides has been employed to identify murine, human and bovine T-cell epitopes for a variety of antigens.Materials and Methods: The strategy of this study was to immunise cattle with recombinant mycobacterial proteins which were known to be dominant bovine T-cell antigens (MPB70, MPB64 and MPB59). From published amino acid sequences, overlapping synthetic peptides incorporating the signal and mature protein of each antigen, were generated as 20-mers with 10 residue overlaps. Peptides were prepared with free N- and C- termini using a BT7400 multiple peptide synthesiser running FMOC chemistry. Individual peptides were semi-purified by C 1 8 Sep Pak methodology and lyophilised from 1% v/v acetic acid. T-cell responses in immunised cattle to individual peptides were screened using the lymphocyte proliferation assay (LPA) and in M. bovis experimentally infected cattle by LPA and IFN-γ production.Results: Eleven mycobacterial epitopes which were recognised by bovine T-cells have been identified in this study: three epitopes for MPB70, five epitopes for MPB64 and three epitopes for MPB59 respectively. These epitopes gave lymphocyte proliferative responses in at least 34 cattle immunised with a cocktail of recombinant antigens. Interestingly, only a proportion of these epitopes were identified as dominant when assayed in animals experimentally infected with M. bovis suggesting that these epitopes are potentially cryptic in the native antigen. Currently these epitopes are being tested for recognition by field cases of bovine tuberculosis to assess their potential as diagnostic reagents.Conclusion: This study has identified bovine T-cell epitopes for MPB70, MPB64 and MPB59 which could form part of a more specific diagnostic test. It is currently our attention to use a cocktail of these peptides in T-cell assays to assess if improved sensitivity and specificity is feasible.