This study was designed to characterize temporal changes of oxidative metabolism in microvascular endothelium in vivo associated with neutrophil adhesion and diapedesis. The rat mesentery was loaded with dichlorofluorescein (DCFH) diacetate, a hydroperoxide-sensitive fluorescent probe that is trapped within viable cells as a nonfluorescent form and is converted to fluorescent dichlorofluorescein (DCF) by hydroperoxides. The fluorescent light emission was recorded with digital microscopy and its camera response was calibrated by superfusion of the mesentery with known concentrations of hydroperoxides. After rinsing with a precursor-free solution, it was possible to observe both the neutrophil adhesion and fluorescence changes in venular endothelium during superfusion with platelet-activating factor (PAF, 100 nM) by alternately changing light sources. In the PAF-treated animals, DCF fluorescence in the venular wall was markedly elevated concurrent with an increase in the number of adherent neutrophils. An onset of a significant DCF fluorescence was observed as early as 10 min after PAF application. The venular oxidative changes were co-localized with neutrophils adhering to the endothelium or migrating into the interstitium. The oxidative impact in the venular wall superfused with 100 nM PAF for 40 min was equivalent to that induced by the 10-min superfusion of 0.8 mM of tert-butyl hydroperoxide. Pretreatment with dipyridyl, an iron chelator, or dimethylthiourea, a hydroxyl radical scavenger, significantly attenuated the PAF-induced oxidative changes in the venule but had little effect on neutrophil adhesion. These findings suggest that an endothelial cell-dependent mechanism involving an iron-catalyzed reaction may have a role in enhancement of neutrophil-mediated oxidative stress in venular endothelium.