BACKGROUND: Adaptive human natural killer (NK) cells are an NK cell subpopulation arising upon cytomegalovirus (CMV) infection. They are characterized by CD94/NKG2C expression, a mature CD57 PURPOSE: We developed a method to efficiently expand adaptive NK cells from NK-enriched cell preparations derived from the peripheral blood of selected CMV-seropositive healthy donors. The method is based on the use of an anti-CD94 monoclonal antibody (mAb) combined with IL-2 or IL-15. RESULTS: By setting this method we were able to expand high numbers of NK cells showing the typical adaptive phenotype, CD94/NKG2C CONCLUSIONS: By this study we provide hints to improve previous expansion methods, by eliminating the use of genetically modified cells as stimulators, and obtaining effectors not expressing unwanted inhibitory receptors. This new protocol for expanding functional adaptive NK cells is safe, cost-effective and easily implementable in a GMP context, suitable for innovative immunotherapeutic purposes.