Natural killer (NK) cells are highly resistant to transfection by conventional methods such as electroporation and lipofection. Recently, we reported the employment of a novel electroporation-based method, called nucleofection, which for the first time enabled efficient nonviral gene transfer into NK cells. In this study, we aimed at developing optimized conditions for the transfection of different NK cell lines as well as primary NK cells. Using EGFP (enhanced green fluorescent protein) or luciferase as reporter genes, suitable buffer conditions as well as instrument settings were defined. The new transfection methodology represents a useful tool for the immunotherapeutic use of NK cells, with the potential to enhance cytotoxicity as well as retarget the specificity of cytotoxic lymphocytes in clinical therapy of cancer and viral infection.