The mycobacterial 65-kDa stress protein has been shown to be a major target for the cellular immune response to pathogenic mycobacteria in humans and animals. T cells reactive with the mycobacterial 65-kDa stress protein have been isolated from infected individuals which may imply a role for T cells in the immunopathogenesis of mycobacterial infections. For these reasons the 65-kDa stress protein of M. paratuberculosis was chosen for the investigation of the immune response to M. paratuberculosis infection in sheep. We have generated recombinant M. paratuberculosis 65-kDa stress protein (rhsp65) and have investigated the response of T cells to this antigen. Peripheral blood mononuclear cells (PBMC) were used to assess T cell responses to rhsp65 in an in vitro proliferation assay. PBMC purified from M. paratuberculosis-infected, rhsp65-primed, CFA-M.75 primed or non-primed animals were cultured in vitro with various concentrations of rhsp65. Flow cytometry was used to characterize the T cell subsets involved in proliferative responses to rhsp65. PBMC from infected and rhsp65-primed animals showed significant proliferative responses to rhsp65 in vitro. No significant proliferative responses were seen by PBMC from CFA-primed or non-primed animals. FACS analysis suggested that CD8+ or gamma/delta T cells may be involved in the in vitro proliferative response to rhsp65. Immunohistochemical analysis was used to investigate the presence of M. paratuberculosis 65-kDa stress protein in infected ileum. An anti-rhsp65 mab reacted with lamina propria cells in infected ileum but not in non-infected ileum. This investigation has demonstrated that paratuberculosis-infected animals have T cells that recognize, and are capable of responding by proliferation to, M. paratuberculosis 65-kDa stress protein in vitro. Furthermore, this protein was present in infected tissue. These preliminary findings support a role for M. paratuberculosis 65-kDa stress protein in the immunopathology associated with ovine paratuberculosis.