Estimation of the oxidised form of glutathione (GSSG) in an ischaemic/reperfused organ is frequently employed as an indicator of oxidative stress created by the production of oxygen free radicals during the reperfusion period. The time course of oxidative stress and tissue damage in 19 ischaemic/reperfused guinea-pig island skin flaps was evaluated. No-flow ischaemia was induced in the flaps for 6 h in 7 animals, and for 8 h in 9 animals (a further 3 animals served as controls without ischaemia). Arterial and venous blood samples were obtained directly from the flap pedicle at baseline, 10, 30, and 60 min following reperfusion. Results suggest that a second focus of oxidative injury, possibly mediated by activated neutrophils, contributes to the overall process of reperfusion injury. Plasma levels of GSSG allow for a more sensitive quantification of oxidant stress within reperfused ischaemic flaps, and may serve as a useful tool in skin flap research.