Both ischemia and reperfusion injury and contractile activity are associated with the 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 before analysis by electron-spin resonance (ESR) techniques. Previous analysis of the ESR signal indicated that the circulating species is either a carbon- or oxygen-centered lipid-derived free radical. The current data indicate that this species is present in the blood of anesthetized rats after 4-h ischemia and 1 h of reperfusion of a single hindlimb. During 4 h of ischemia, the species was also present in microdialysates from the tibialis anterior muscle but was unchanged in magnitude compared with control tissue. During 1 h of reperfusion, the signal intensity increased by a mean of 420% (P < 0.05, n = 4). Hydroxyl radical activity in the interstitial fluid also significantly increased during ischemia and further increased by a mean of 210% (P < 0.05, n = 4) during reperfusion. No changes in interstitial superoxide levels were seen, but interstitial PGE₂ content also increased during reperfusion. A significant positive correlation was found between the magnitude of the ESR signal and both the hydroxyl radical activity and PGE₂ content of microdialysis fluids. These data support the hypothesis that the circulating free radical species is formed in the interstitial fluid by hydroxyl radical interaction with a lipid that may be released from reperfused tissue with a similar pattern to prostanoids.