We have developed a technique for analysis of granulocyte reactive oxygen species formation in whole blood using flow cytometry and two color immunofluorescence. This technique relies upon the use of specific fluorescent dye (LDS-751) to stain nucleated cells, eliminating erythrocytes from analysis. Using LDS-751, forward angle light scatter, and 90 degrees side scatter, a granulocyte gate, monocyte gate, and lymphocyte gate were identified. Analysis with multiple FITC conjugated monoclonal antibodies demonstrated greater than 95% purity of a flow cytometrically identified granulocyte population in whole blood without physical manipulation of the blood. Utilizing 2'7' dichlorofluorescein diacetate (DCFH-DA), we were able to measure granulocyte intracellular reactive oxygen species production. Dose response curves were obtained for the effect of granulocyte agonists phorbol myristate acetate, FMLP, and heat fixed Staphylococcus aureus on reactive oxygen species production. The techniques described in this paper should be useful for measuring granulocyte activation in vivo with flow cytometry.