To test whether exogenous oxidants alter intracellular oxidant levels in skeletal muscle fibres, we exposed rat diaphragm to donors of nitric oxide (NOx), reactive oxygen species (ROS) or hyperoxia, and monitored intracellular oxidant levels using a fluorescent probe. Fibre bundles were dissected from the diaphragm and loaded with 2’,7’-dichlorodihydrofluorescein (DCFH); emissions were monitored using a fluorescence microscope. DCFH-loaded muscles were exposed to either a NOx donor (1 m M S-nitroso-N-acetyl penicillamine, SNAP; 1 m M sodium nitroprusside, SNP; 400 μM 1-hydroxy-2-oxo-3-(N-3-methyl-aminopropyl)-3-methyl-1-triazen, NOC-7), an ROS donor (100 μM hydrogen peroxide, H2O2; 100 μM tert-butyl hydroperoxide; 1 m M hypoxanthine plus 0.01 U mL–1 xanthine oxidase, HXXO) or a range of PO2s (25, 60 or 95% O2 oxygenating Krebs–Ringer solution) for 40 min; time-matched control bundles remained in Krebs–Ringer solution. Control muscles oxidized DCFH at a rate of 0.32 ± 0.1 greyscale units min–1. SNAP (766%), SNP (1244%), NOC-7 (851%), H2O2 (543%), and HXXO (541%) increased DCFH oxidation from control levels. The increase in emissions caused by NOC-7 and SNP were blunted by the NOx scavenger haemoglobin (1 μM). DCFH oxidation by HXXO was unaffected by 1000 U mL–1 superoxide dismutase but was significantly decreased by 1000 U mL–1 catalase and 1 m M salicylate. PO2 had no effect on intracellular oxidant levels. Therefore, extracellular NOx and ROS can alter intracellular oxidant status in skeletal muscle fibres. These observations suggest that intrafibre oxidant levels could be the result of both intracellular and extracellular oxidant production.