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Nonmyeloablative allogeneic stem-cell transplantation (alloNST) is the focus of investigations searching for less-toxic transplantation regimens. We report studies on the kinetics of lymphodepletion and safety of pentostatin (PT) conditioning in alloNST. Patients with hematologic malignancy received mobilized blood from human leukocyte antigen-matched related (n=4) or unrelated (n=8) donors. PT 4 mg/m2 was administered on days −21, −20, and −19 and 200 cGy of total-body irradiation was administered on day −1, followed by cyclosporine A and mycophenolate mofetil. Mononuclear cell adenosine deaminase after PT was inhibited 84%. The absolute CD3+ cells decreased significantly by day −7 (49%) and CD19+ cells declined 92% by day −1. CD4+ cells were depressed more than CD8+ cells. Neutrophils and monocytes were minimally affected by PT. Median posttransplant peripheral blood chimerism on day 70 showed 95% donor leukocytes and 82.5% donor CD3 lymphocytes. PT demonstrated lymphodepleting effects and promising safety, supporting alloNST as early as 7 days after initiation of PT.