Previously reported observations have indicated an association between increased numbers of Vδ1+ γδ T cells and disease free survival after partially mismatched bone marrow transplantation, suggesting a graft versus leukemia effect. Studying the interactions between such cells and leukemic target cells has proven difficult since cells expressing γδ forms of the T cell receptor (TCR) comprise a very small percentage of circulating T cells and are not easily expanded in vitro. We have, therefore, developed an in vitro culture system in which γδ T cells derived from bone marrow donors can be expanded in the presence of leukemic cells, i.e. under conditions similar to those found in partially mismatched transplant recipients, in order to allow their phenotypic and functional characteristics to be studied in detail. Donor recipient pairs were selected for these experiments using pre-transplant candidates in relapse status. Donor mononuclear cells obtained from peripheral blood were depleted of αβ T cells using anti-CD4 and anti-CD8 immunomagnetic microspheres. Recipient blast cells were obtained from peripheral blood by Ficoll separation, followed by Percoll gradient centrifugation to remove other hematopoietic cells, and were irradiated immediately prior to plating. Donor cells depleted of αβ T cells, and irradiated recipient blast cells were then co-cultured both in the presence and absence of an anti-TCR yo monoclonal antibody previously shown to stimulate a limited expansion of y5 T cells in vitro. Donor cells co-cultured with recipient blasts alone failed to proliferate. However, in the combined presence of both antibody and blast cells, donor cells underwent a vigorous expansion characterized by a 1000-2000 fold increase in the number of Vδγ1+ γδ T cells. Furthermore, the phenotype of these cells closely resembled that of y5 T cells isolated from transplant recipients with increased numbers of γδ T cells. By contrast, although donor cells exposed to antibody alone underwent a similar expansion, they were found to evolve an almost entirely Vδ2+ phenotype. These results suggest that populations of cells very similar to those observed clinically can be generated in vitro. Future experiments will determine the potential of such cells to mediate a direct graft versus leukemia effect.