Abstract The activity of eosinophil peroxidase (EPO) is commonly employed as a measure of eosinophil activation in biologic fluids. Determination of product formation by this enzyme by end-point measurement may be affected profoundly by substrate concentrations, reaction time and degradation of end-product and enzyme. To determine more accurately EPO concentrations in media conditioned by isolated, purified eosinophils, we have developed a kinetic, colorimetric assay to measure EPO concentration as a function of maximum velocity of reaction ( V max). An automated method for determining V max in a 96-well microplate colorimetric assay was utilized over a wide range of substrate concentrations. Concentrations ⩾3 × 10 −8 g/ml could be determined reiably with this assay. Peroxidase activiy was inhibited in a concentration-dependent manner by the addition of 3-amino-1,2,4-triazole (AMT). The EPO concentration in eosinophils determined by this kinetic method was ∼ 1.1 × 10 −5 g/10 6 eosinophils. Eosinophil activation with 10 −6 M f-Met-Leu-Phe (fMLP) caused substantial EPO secretion (9.0 ± 1.7% vs.2.9 ± 0.6% total EPO content for control, P = 0.05) and decrease in eosinophil EPO concentration (92.3 ± 4.2% of control, P = 0.038). Secretion was enhanced by the addition of 5 μg/ml cytochalasin B to 10 −6 M fMLP (25.9 ± 12.7% total EPO content, P = 0.043 vs. control); similar decreases were noted in eosinophil EPO concentration (71.7 ± 16.1% of control, P = 0.043). These data demonstrate that determination of EPO secretion by measurement of V max is a reliable, accurate method for precise quantification of this enzyme in media containing purified eosinophils or eosinophil products in the absence of other forms of peroxidase activity.