A rapid and sensitive method is described for the quantitative determination of H202 produced by phagocytizing human granulocytes. For this purpose, the method of Keston and Brandt was mechanized, which is based on the oxidation of nonfluorescent leukociacetyl-2,7-dichlorofluorescein to a fluorescent compound by H202 in the presence of peroxidase. The optimal conditions for this test were determined. H202 in water can be measured in the range of 0.05 to 0.5 muM, with a standard deviation of 1.2 per cent at 0.4 muM (n = 1-). The production of H2O2 by phagocytizing granulocytes could only be measured in a medium which contained phosphate-buffered salt, albumin, glucose, NaN3, and IgG-coated latex particles. The fluorescence signal was catalase-sensitive. Of known amounts of H202, added to this medium, 97 per cent were recovered. Under optimal conditions we found a H2O2 production of 970 plus or minus 170 mumoles per 10-10 cells per hour (10 different healthy donors), corresponding to 50 to 70 per cent of the observed increase in O2 consumption. No H2O2 was produced by phagocytizing granulocytes from 2 patients with chronic granulomatous disease, while intermediate values were found in the cells from heterozygotes.