BACKGROUND The use of 2',7'-dichlorofluorescein diacetate (DCFH), dihydrorhodamine 123 (DHR) and hydroethidine (HE) has been described for detecting respiratory burst activity by flow cytometry in polymorphonuclear neutrophil (PMN) suspension. However, their specificities for reactive oxygen species are not well defined. We investigated the reactivity of these probes for detecting superoxide anion (O(2)(* -)), hydrogen peroxide (H(2)O(2)) and/or nitric oxide (NO(z.rad;))-dependent mechanisms. METHODS PMNs (10(6)/ml) were preincubated for 15 min at 37 degrees C with DCFH (5 micro mol/l), DHR (1 micro mol/l) or HE (10 micro mol/l). Cell suspensions were then split for each probe into five different aliquots containing either no effector or one effector: N-ethylmaleimide (NEM, 150 micro mol/l, NADPH oxidase inhibitor), sodium azide (NaN(3), 50 micro mol/l, peroxidase and catalase inhibitor), N-nitro-L-arginine methyl ester (L-NAME, 1.5 micro mol/l, NO(z.rad;) synthase inhibitor) or H(2)O(2) (30%). At the same time, PMNs were stimulated with phorbol myristate acetate (PMA, 10 micro mol/l) for 10 min at 37 degrees C. Analyses were carried out on a Beckman-Coulter Epics XL equipped with an argon laser (488 nm). Green fluorescences from DCFH and DHR were measured in the FL1 channel and HE fluorescence was analyzed in the FL2 channel. RESULTS NaN(3) decreased the fluorescence of PMNs incubated with DCFH, indicating that it needs a peroxidase activity to react with H(2)O(2). L-NAME reduced the oxidation of DCFH, showing that it reacts with reactive nitrogen species. DHR was specifically responsive to H(2)O(2) accumulation. HE seemed to be preferentially oxidized by O(2)(* -). CONCLUSIONS Hence the choice of the probe to be used depends on the reactive species of interest.