This pilot study tested feasibility of natural killer cell purification and infusion (NK-DLI) in patients after haploidentical hematopoietic stem cell transplantation (HSCT). The aim was to obtain >or=1.0 x 10(7)/kg CD56+/CD3- NK cells and <1.0 x 10(5)/kg CD3+ T cells. Mononuclear cells were collected by 10 l leukapheresis. A two-step ex vivo procedure was used to purify NK cells, using an immunomagnetic T-cell depletion, followed by NK-cell enrichment. Five patients with high-risk myeloid malignancies were included, presenting 3-12 months after a haploidentical HSCT with mixed chimerism (3), impending graft failure (1) or early relapse (1). The purified product contained a median of 1.61 x 10(7)/kg (range 0.21-2.2) NK cells and 0.29 x 10(5)/kg (0.11-1.1) T cells. A purity of NK cells of 97% (78-99), a recovery of 35.5% (13-75), and a T-cell depletion of 3.55 log (2.9-4.5) was achieved. Infusions were well tolerated and none of the patients developed graft-versus-host disease. We observed an increase in donor chimerism in 2/5, stable mixed chimerism, decreasing chimerism and relapse of AML in one patient each. Selection of NK-DLI is technically feasible. NK cells are well tolerated when used as adoptive immunotherapy in recipients of haploidentical HSCT.