The release of hydrogen peroxide from human blood platelets after stimulation with particulate membrane-perturbing agents has been determined by fluorescence using scopoletin as the detecting agent. Platelet suspensions containing less than 1 polymorphonuclear leukocyte/10(8) platelets showed a significant release of hydrogen peroxide (6.11 nmol/10(9) platelets per 20 min, S.D., 0.26, n = 9) after addition of zymosan or latex particles, compared to unstimulated platelets. The release of hydrogen peroxide was only observed when the scopoletin was added to the platelet suspensions during the stimulation. Any attempt to determine hydrogen peroxide release in the supernatant at the end of the incubation with zymosan or latex failed. A NADH-dependent production of hydrogen peroxide was observed by measuring the difference of oxygen uptake in the presence and absence of catalase (500 units), which was not inhibited by potassium cyanide (1 mM). By this method the NADH-dependent cyanide-insensitive peroxide production and release was 6.0 nmol/10(9) platelets per 20 min from resting platelets (S.D., 2, n = 6) vs 15 nmol/10(9) platelets per 20 min from stimulated platelets (S.D., 2, n = 6).