Reactive oxygen species (ROS) have been shown to participate in tissue injury after ischemia-reperfusion. Their implication in leukocyte adherence and increase in permeability at the venular side of the microcirculation has been reported, but very little is known about ROS production in arterioles. The objective of this work was to evaluate, in the arteriole wall in vivo, the temporal changes in superoxide anion production during ischemia and reperfusion and to identify the source of this production. Mouse cremaster muscle was exposed to 1-hour-ischemia followed by 30-min of reperfusion, while superoxide anion production was assessed by a fluorescent probe, i.e. intracellular dihydroethidine (DHE) oxidation. During ischemia, we found a significant increase in DHE oxidation and no additional increase in fluorescence was observed during the following reperfusion. This phenomenon was significantly inhibited by pre-treatment with superoxide dismutase. Allopurinol (xanthine oxidase inhibitor) or stigmatellin (Q_o-site inhibitor of mitochondrial complex III) or simultaneous administration of these two inhibitors significantly reduced superoxide production during ischemia to 80%, 88% and 72% of that measured in untreated I/R group. By contrast, no significant inhibition was found when NADPH oxidase was inhibited by apocynin or when mitochondrial complex I or complex II were inhibited by rotenone or thenoyltrifluoroacetone. A significant increase in ROS was found with Antimycin A (Q_i-site inhibitor of mitochondrial complex III). We conclude that significant increase in ROS production occurs during ischemia in the arteriolar wall. This increased production involves both a cytoplasmic source (i.e xanthine oxidase) and the mitochondrial complex III at the Q_o-site.