Bluetongue virus (BTV), an arbovirus transmitted by midges, can cause serious disease in sheep. Both virus neutralizing antibody and cytotoxic T lymphocytes (CTL) have been shown to have a role in protective immunity. In this study, the antigen specificity of CTL from BTV-immune sheep has been determined using recombinant vaccinia viruses expressing individual BTV antigens. The results show that, in the sheep studied thus far, the serotype-specific outer coat protein, VP2, and the non-structural protein, NS1 are major immunogens for CTL, with VP5 (an outer coat protein) and NS3 being minor immunogens. No VP7 (a major group-reactive inner coat protein) specific CTL were detected. The CTL from sheep immunized with serotype 1 were cross-reactive and able to recognize target cells infected with other BTV serotypes. Further work demonstrated that the cross-reactive CTL recognized NS1, but not VP2.