To allow differentiation between the pathological and physiological role that ROS play in skeletal muscle, it is essential to identify and quantify these species. We have previously demonstrated increased superoxide (O₂^.–) and hydroxyl (^null.nullOH) production in muscle extracellular space during exercise (McArdle et al., 2001;2004). These methods involved the assumption that O₂^.– reduces cytochrome c (CC) and that ^.OH causes the hydroxylation of salicylate; however, nitric oxide (NO) and peroxynitrite (ONOO^–) may also contribute to these processes (Murrant and Reid, 2001). This study examined the effects of specific ROS inhibitors on the reduction of CC and hydroxylation of salicylate in vivo using microdialysis. 24 male C57Bl/6 mice were allocated into 4 groups. Group 1 received an IV injection of 50mg/kg b.wt of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). Group 2 received 10mg/kg of the iron chelator desferrioxamine mesylate (desferal) which prevents the formation of null;OH via Fenton chemistry, and group 3 received 5,000U/kg superoxide dismutase (SOD), which catalyses the dismutation of O₂^.–, both by IP injection. Group 4 were untreated controls. 30-min post drug administration, mice were anaesthetised with sodium pentobarbitone (5-10mg/100g IP) and supplemental doses were given as required to maintain a sufficient depth of anaesthesia. Whilst anaesthetised, 2 microdialysis probes were placed into the gastrocnemius muscle and perfused with salicylate or CC in saline. O₂^.– activity was analysed in the perfusates by measurement of CC reduction and ^.OH activity by measurement of 2,3-dihydroxybenzoate (2,3-DHB) formed from salicylate (McArdle et al., 2004). Following 1 hr of baseline collections, the hind-limb was subjected to a 15-min period of isometric contractions (McArdle et al., 2001). Mice were humanely killed 30 min post-exercise. Data was analysed using a mixed design ANOVA (P<0.05). The reduction in CC was greater in mice treated with L-NAME compared with control mice (e.g. mean resting value = 0.34 (SD 0.03) versus 0.25 (SD 0.09) nmol/15 min, P<0.05). The reduction of CC was attenuated in mice treated with SOD compared with control mice, (e.g. mean resting value = 0.16 (SD 0.02) versus 0.25 (SD 0.09) nmol/15 min, P<0.05). The hydroxylation of salicylate was attenuated in mice treated with desferal compared with control mice (e.g. mean resting value = 3.77 (SD 2.81) versus 8.28 (SD 2.50) pmol/15 min, P0.05). Data suggest that O₂^.– is the major contributor to the reduction of CC and ^.OH causes the hydroxylation of salicylate, supporting the use of these assays to detect extracellular ROS generation in vivo using microdialyis.