This study tested the hypothesis that exercise-induced oxidative stress is caused by free radical-mediated damage topolyunsaturated fatty acids (PUFA) which can be prevented following ascorbate prophylaxis. Hyperfine coupling constants(HCC) of a-phenyl-tert-butylnitrone (PBN)-adducts were measured via room temperature electron paramagnetic resonance(EPR) spectroscopy in the venous blood of 12 subjects at rest and following maximal exercise during a randomized doubleblindplacebo-controlled trial and compared to those observed following room-air incubation (2 h at 378C) of L aphosphatidycholine,linoleic acid, a-linolenic acid and arachidonic acid. All adducts exhibited similar HCC [aN 13.6 Gauss(G) and abH 1.8 G] with the exception of L-a-phosphatidycholine [aN113.4 G, abH11.6 G (37%) and aN214.9 G,abH20.3 G (63%)] consistent with the trapping of lipid-derived alkoxyl and oleate radicals, respectively. Ascorbate pretreatmentablated radical formation in both systems. These findings identify circulating PUFA as a potential source ofsecondary radicals that are capable of initiating oxidative stress in the exercising human.