Mycobacterium bovis infection in cattle continues to be a problem in several regions, partly due to inadequate diagnostic tests. The aim of this study was to use an experimental model of the natural disease to identify T-cell epitopes from the mycobacterial 38 kDa antigen as potentially specific diagnostic reagents. A panel of overlapping synthetic peptides (16-mers with a five-residue overlap) were produced from the published amino acid sequence. It was found that peripheral blood lymphocytes from at least three of four experimentally infected animals, which were considered to be in either Th1- or Th1/Th2-dominated stages of anti-mycobacterial immunity, proliferated in response to five epitopes (residues 1-27, 88-107, 122-138, 243-260 and 307-328). However, in vitro production of IFN-gamma was detected only in response to epitope 122-138, indicating a role in protective immunity. The peptides were not recognized by control, uninfected animals, but all epitopes showed various degrees of recognition by animals which were field reactors to intradermal tuberculin testing. Furthermore, epitopes 1-27, 88-107 and 122-138 were recognized by four breeds of cattle and by animals from separate herds, suggesting genetic permissiveness in recognition which would be essential in the development of a diagnostic test.