Both ischaemia and reperfusion injury and contractile activity are associated with generation of reactive oxygen species and free radicals by skeletal muscle. In addition exercise has been reported to lead to the formation of a circulating free radical species that is detectable in the blood by spin trapping prior to analysis by electron spin resonance (ESR) techniques. Previous analysis of this signal has suggested that the circulating species is either a carbon or oxygen-centered lipid derived radical. The data presented here indicate that this species is present in the blood of anaesthetised rats following prolonged (4 hours) ischemia and 1 hour reperfusion of a single hind limb. Microdialysis studies demonstrated that the species was present in the interstitial fluid of the tibialis anterior muscle. During 4 hours ischaemia the magnitude of the ESR signal from microdialysates was unchanged, but during 1 hour reperfusion the signal intensity increased by a mean of 420% (P<0.05; n=4). Hydroxyl radical activity in the interstital fluid was found to increase significantly during the ischaemic period and to further increase by a mean of 210% (P<0.05; n=4) during reperfusion. No changes in interstitial superoxide levels were seen, but interstitial PGE₂ was found to increase during reperfusion. Comparison of the pattern and magnitude of the ESR-detectable species in microdialysates with measures of extracellular superoxide, hydroxyl radical and prostaglandin E₂ release into the interstitial fluid revealed a significant positive correlation between the magnitude of the the ESR signal and both the hydroxyl radical activity and PGE₂ content. These data support the hypothesis that the circulating free radical species is formed by hydroxyl radical interaction with a lipid within or adjacent to the muscle interstitial space, and that the lipid involved may be released from the reperfused tissue with a similar pattern to prostanoids