Natural killer (NK) cells are thought to be of benefit in HLA-mismatched hematopoietic transplantation (H-SCT). Therefore, we developed a protocol for clinical-use expansion of highly enriched and IL-2-stimulated NK cells. Purification of unstimulated leukaphereses by a two-step T cell depletion with a final CD56 enrichment procedure leads to a mean purity of 95% CD56(+)CD3- NK cells with a four- to five-log depletion of T cells. So far, three pediatric patients with multiply relapsed acute lymphoblastic leukemia (ALL) or acute myelogenous leukemia (AML) were treated with repeated transfusions post-H-SCT. Directed killer immunoglobulin-like receptor (KIR) mismatches were demonstrated in all three cases. Although all patients showed blast persistence at the time of transplant, they reached complete remission and complete donor chimerism within 1 month post-H-SCT. NK cell therapy was tolerated well without graft-versus-host disease (GvHD) induction or other adverse events. The AML patient died of early relapse on day +80, while the ALL patients died of thrombotic-thrombocytopenic purpura and atypical viral pneumonia on days +45 and +152, respectively. This initial trial showed the feasibility of good manufacturing practice (GMP)-compliant NK cell isolation and expansion for clinical applications. We now launch a clinical phase I trial with activated NK cells post-H-SCT.