Objective: To develop a simple, flow cytometric assessment of Natural Killer (NK) cell activation.Introduction: The current method used to analyze NK-cell function is the chromium release assay. This method has disadvantages, including the use of radioactive materials and maintaining a target cell line. This study investigates a novel flow cytometry assay that measures NK-activity using heat shock protein-60 (HSP60) induced CD69 expression, while eliminating the need for radioactive materials, and target cell culture.Methods: Heparinized blood was obtained from 25 healthy volunteers. To activate the NK-cells, 500 μl of whole blood was incubated with 50 μl of 12.5 μg/ml HSP60 overnight. 100 μl of each sample was then labeled with anti-CD56, anti-CD45 and anti-CD69 fluorescent conjugated mAb and analyzed on a Becton Dickinson FACs calibur cytometer. 10,000 events were collected from each tube. The percent of CD56, CD69+ lymphocytes was determined. We also determined NK-cytotoxicity against K562 target cells for each specimen using a standard 4-hour chromium release assay. To validate the flow cytometry assay, we performed a linear regression analysis to determine if HSP60 induced CD69 expression correlated with the chromium release assay.Results: HSP60 induced CD69 expression on a mean of 36.5% (Range = 9.4%–67.0%) of NK-cells. Regression analysis of %CD69+ NK-cells and % lysis from the chromium release assay showed no linear relationship between the 2 sets of data (r squared = 0.042). NK% varied considerably among our volunteers (Range = 4.3%–23.7%). Thus, we determined a unit of activation ((%NK-cells × %CD69+ NK-cells)/100) to control for differences in NK numbers and repeated the regression analysis. The r-squared value was 0.2739 using the unit of activation. Semi-quantitative analysis of this graph, however, indicated that there are four clusters within the data corresponding to high, medium, medium-low, and low ranges of CD69 expression corresponding to four similar ranges of NK lytic activity.Conclusion: This study shows that HSP60 induced CD69 expression measured by flow cytometry can be used as a semi-quantitative surrogate marker for NK-cell activity. This assay eliminates risks involved with using radioactive materials, and also cuts down on preparation and analysis time for lab personnel. Further studies will show if this assay has clinical applicability.We thank the Summa Health System Foundation for supporting this study and Dr. Steven Douglas for providing the K562 target cell line.