Pairs of murine monoclonal antibodies, which recognize 2 different epitopes on a single antigen are described. These antibodies (MdT-P1, -P2, -Q1, -Q2) defining a canine pan-T cell antigen, were raised against dog thymocytes. In immunoblotting of solubilized and polyacrylamide gradient gel electrophoresis in sodium dodecyl sulphate (SDS-PAGE) fractionated dog thymocytes, they revealed a strong heterogeneous antigen. Competitive inhibition of binding of directly labeled mouse-antidog T lymphocytes monoclonal antibodies (MdT-mAbs) to solubilized dog thymocytes indicates that 2 different antigenic epitopes (P, Q) are recognized. In indirect peroxidase immunocytochemistry, MdT monoclonal antibodies recognized up to 95% thymocytes, 69% blood lymphocytes, 76% lymph node lymphocytes, and approximately 2% bone marrow lymphocytes; they were nonreactive with surface immunoglobulin positive blood cells, monocytes, platelets, cells of myelo- and erythropoietic lineage in the bone marrow. Immunohistochemistry on thymus, lymph nodes, and spleen sections revealed that MdT-mAbs had labeled cortical and medullary thymocytes, paracortical T cell areas in lymph nodes and the periarteriolar zone of spleen white pulp, whereas B cell areas remained unstained. The antibodies lysed dog thymocytes in the presence of complement. Lethally irradiated dog receiving bone marrow autograft depleted of MdT-P1 positive cells ex vivo showed engraftment and complete recovery of marrow function. Studies of antibody activity on canine hemopoietic progenitor cells in granulocyte-macrophage progenitors (CFUGM) also showed no reduction of CFUGM in MdT-P1-depleted bone marrow.