Recently, the Histiocyte Society revised the diagnostic criteria for hemophagocytic lymphohistiocytosis (HLH) to include low or absent natural killer (NK) cell activity, according to local laboratory reference. The aim of this study was to establish reference interval for functional NK-cell activity in 63 healthy Korean individuals using a flow-cytometric assay. We used peripheral blood mononuclear cells (PBMCs) as effector cells and Fluorescein isothiocyanate-labeled K562 cells as target cells. NK-cell activity was calculated using the following equation: NK-cell activity (%) = (test lysis - spontaneous lysis) x 100/(maximum lysis - spontaneous lysis). NK-cell activity was analyzed in 13 known HLH patients and 16 suspected non-HLH patients using a flow-cytometric assay. The mean (+/-SD) cytotoxicity of PBMCs from healthy individuals was 20.9 +/- 5.3% and the reference interval was 11.8-31.9%. The mean NK-cell activity of HLH patients (8.3 +/- 8.9%) was significantly lower (P = 0.001) than that of non-HLH patients (20.1 +/- 7.8%). The sequential changes in NK-cell activity in the HLH group corresponded to clinical and laboratory findings following treatment. We successfully developed a functional NK-cell activity test for use in the clinical laboratory and obtained a reference interval of NK-cell activity from healthy donors. This assay, and associated reference interval, was used to analyze 30 clinically relevant specimens and the results were shown to be well correlated.