Adoptive immunotherapy using allogeneic natural killer (NK) cells provides to be useful in recipients after allogeneic hematopoietic stem cell transplantation (Allo-HSCT), but its application has been limited by the inability to obtain sufficient numbers of pure NK cells. This study was aimed to optimize the expansion of high purity NK cells from human peripheral blood. First, the NK cells were isolated from PBMNC by using miniMACS (magnetic cell-selection) and NK Cell Isolation Kit II. Then the isolated cells were cultured in SCEM (Stemline Hematopoietic Stem Cell Expansion Medium, Sigma) supplemented with 10% human AB serum and different combinations of IL-2 and/or IL-12, IL-15 for 15 days. Cultures were fed with fresh media and cytokines every 3 days, and were evaluated for cell expansion, phenotype, and cytotoxicity at the end of the culture period. The results showed that in group IL2 + IL15 and IL2 + IL15 + IL12, cells were expanded 50.46 +/- 4.31 and 52.35 +/- 6.72-fold respectively, much higher than others (P<0.01), but no significant difference between themselves (P>0.05). And the purity of CD3(-)CD56(+) NK cells was over 94% in all groups except the control. The cytotoxicity of expanded NK cells cultured with cytokines was significantly higher than the starting population at different E:T ratio (P<0.01), although the cytotoxicity of IL2 + IL15 + IL12 group was slightly higher than that of IL2 + IL15 group, but no significant difference between themselves (P>0.05). It is concluded that high purity of NK cells can be efficiently expanded in culture with IL2 + IL15.