Purpose: To determine the sensitivity of the 18F-radiolabelled dihydroethidine analogue ([ 18F]DHE) to ROS in a validated ex vivo model of tissue oxidative stress. Procedures: The sensitivity of [ 18F]DHE to various ROS-generating systems was first established in vitro. Then, isolated rat hearts were perfused under constant flow, with contractile function monitored by intraventricular balloon. Cardiac uptake of infused [ 18F]DHE (50–150 kBq.min −1) was monitored by γ-detection, while ROS generation was invoked by menadione infusion (0, 10, or 50 μm), validated by parallel measures of cardiac oxidative stress. Results: [ 18F]DHE was most sensitive to oxidation by superoxide and hydroxyl radicals. Normalised [ 18F]DHE uptake was significantly greater in menadione-treated hearts (1.44 ± 0.27) versus control (0.81 ± 0.07) (p < 0.05, n = 4/group), associated with concomitant cardiac contractile dysfunction, glutathione depletion, and PKG1α dimerisation. Conclusion: [ 18F]DHE reports on ROS in a validated model of oxidative stress where perfusion (and tracer delivery) is unlikely to impact its pharmacokinetics.