Stimulated neutrophils (PMNs) produce large quantities of superoxide anion, which is the precursor for hydrogen peroxide (H2O2). We developed a new fluorimetric assay to measure the H2O2 released by zymosan A-activated PMNs utilizing the oxidation of p-hydroxyphenylacetic acid by H2O2 to its fluorescent dimer in the presence of horseradish peroxidase. Zymosan-activated PMNs isolated from nine healthy volunteers and 20 patients with acute hypoxemic respiratory failure (AHRF) released after 90 min 2.3 +/- 0.3 and 2.4 +/- 1.3 nmol H2O2/10(6) PMNs, respectively. Inhibition of the heme enzymes by 1.0 mM sodium azide (NaN3) increased the H2O2 production to 21.6 +/- 4.4 nmol H2O2/10(6) PMNs in the control group (P less than 0.001), and to 22.5 +/- 14.7 nmol H2O2/10(6) PMNs in patients with AHRF (P less than 0.001). Incubation temperature, room temperature or 37 degrees C, did not change the total amount of H2O2 produced after 90 min by zymosan-activated PMN. Addition of NaN3 improved both the sensitivity and reproducibility of the measurement of H2O2 and allowed detection of H2O2 released by PMNs with coefficients of variation of less than 5% at PMN concentrations as low as 0.1 x 10(6) cells/ml. The amount of H2O2 released by activated PMNs did not distinguish healthy controls from patients with AHRF.