Since CD8+ T cells play an important role in resistance to infection with heartwater, effective vaccines against this disease will likely require identification of antigens that contain CD8+ T cell epitopes responsible for cytotoxic T lymphocyte (CTL) responses. With the use of the fluorescent antigen-transfected target cell (FATT)-CTL assay, IFN-γ ELISPOT and flow cytometry, peptides that induce CTL, proliferation of CD8 + T cells and IFN-γ production were identified as possible target antigens for vaccine development. Of particular relevance was the finding that different peptides from different antigens were able to elicit varied cytotoxic activities by immune peripheral blood mononuclear cells (PBMC) from heartwater immune tick-infected sheep. Several peptides derived from Erum0660, Erum2330, Erum2540, Erum2580 and Erum5000 induced CTL in immune sheep PBMC. Peptide Erum2540-6 was the only peptide that induced significant CTL, CD8+CD45RO+ and CD8+IFN-γ+ by PBMC from all three sheep, and Erum2540 and p2540-20 induced the highest % CTL response in all three outbred sheep. These results suggest that these epitopes may be of major importance in heartwater recombinant vaccine development.