Donor-derived CD4+ T cells may play a role in the development of graft- versus-host disease (GVHD) and graft-versus-leukemia reaction after allogeneic bone marrow transplantation (BMT). Therefore, we evaluated the effect of CD4+ T-cell depletion on GVHD and graft-versus-leukemia reaction after HLA-matched BMT. CD4 depletion was performed using anti-CD4 monoclonal antibodies and immunomagnetic beads, initially in small-scale experiments on bone marrow and granulocyte colony-stimulating factor-mobilized peripheral blood apheresis products. The result was elimination of the CD4+ T cells from both sources (0% and 2 ± 1.4% CD4+ cells, respectively). Subsequently, we used this technique for large-scale negative selection of CD4+ T cells from bone marrow grafts of four consenting leukemic patients in relapse (ALL- 3, ANLL-1) (M-3, F-l). The large-scale CD4+ T-cell depletion resulted in >98% (n=4) elimination of CD4+ cells. The resulting population included 17.7 ± 4.6% CD3+ T cells, 8.9 ± 2.5% CD8+ T cells, 0.1 ± 0.1% CD16+ natural killer cells, and 2.3 ± 3.2% CD34+ hematopoietic progenitor cells. Patients were transplanted with 2.84 ± 1.31x108 viable cells/kg. They received cyclosporine starting on day -1 as GVHD prophylaxis. Engraftment was fast with a white blood cell count of >1x109/L on day 13.2 ± 0.5, an absolute neutrophil count of >0.5X109/L on day 13.8 ± 0.5, and a platelet count of >25x109/L on day 26.5 ± 6.8. Immunological reconstitution was normal, and peripheral blood phenotyping 3 weeks after BMT disclosed 49.0 ± 5.0% CD3, 14.3 ± 12.4% CD4, and 59.5 ± 7.8% CD8 T cells in addition to 17.0 ± 3.0% CD16+ and 9.0 ± 3.0% CD56 natural killer cells. Three out of four patients developed very early grade IV GVHD beginning on day 12 (10-13) and died 2-4 months after BMT. One patient is alive and well with a follow-up of 36 months. We conclude that selective CD4 T-cell depletion does not prevent GVHD.