L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) is a lysosomotropic agent that selectively kills cytotoxic T cells and their precursors, natural killer cells, and monocytes but not helper T cells or other cells of hematopoietic origin. In this study, the effects of treatment of bone marrow and peripheral blood buffy coat with Leu-Leu-OMe on the outcome of allogeneic marrow transplantation were studied in several canine models. Whereas incubation of autologous marrow with Leu-Leu-OMe had no adverse effects on subsequent engraftment, incubation of marrow from dog leukocyte antigen (DLA)-identical littermates resulted in a high rate of graft failure. Previous studies have demonstrated that the addition of peripheral blood buffy coat allows engraftment of unrelated DLA-nonidentical marrow, and in this study we found that incubation of buffy coat with Leu-Leu-OMe did not alter this graft promoting effect. In a final experiment it was demonstrated that incubation of both marrow and peripheral blood buffy coat did not prevent the development of graft-versus-host disease in recipients of marrow from DLA-haploidentical littermates. In considering the eventual application of Leu-Leu-OMe in the clinic, these results are less encouraging than those previously reported using murine models.