Modulation of intracellular signaling pathways or receptor expression in natural killer (NK) cells by genetic manipulation is an attractive possibility in studies of NK cell specificity and function. Moreover, feasible applications of these genetic manipulations in the context of gene and NK cell therapy regimens may be considered. However, efficient gene modification of primary NK cells has been largely hampered by the absence of an efficient gene-transfer protocol.A retrovirus-based easy-to-use transduction protocol that can insert the gene of interest permanently into primary NK cells would be an important tool to advance our studies in NK cell biology and NK cell-mediated therapies. We have recently described a protocol for efficient expansion of NK cells under good manufacturing practice (GMP) conditions from the healthy donors and from patients with hematological malignancies. As the active division of cells is a prerequisite for efficient retroviral insertion, the high rate of expansion in this protocol provides more efficient transduction by retroviral vectors. We hereby present this simple and efficient retroviral vector-based gene-transfer protocol for such ex vivo cultured primary human NK cells.