Introduction: Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are continuously produced by skeletal muscle. Their generation is augmented during contractile activity and may play an important role in signaling adaptive responses and/or mediate some degenerative processes [1]. Purpose: To elucidate the roles of ROS and RNS in detail, we examined their intracellular activities using fluorescence microscopy in single isolated mature skeletal muscle fibres from mice overexpressing neuronal nitric oxide (nNOS) and wild type control mice both at rest and following a period of contractile activity. Methods: Genetically modified mice overexpressing nNOS (type I NOS) [2] and wild type (C57Bl/6) mice were used in this study. Each group consisted of 9 mice aged 22 ± 1 months. Mice were killed by cervical dislocation and the flexor digitorum brevis muscles were dissected. Muscles were incubated in collagenase to isolate single muscle fibres. Fibres were then plated on culture dishes which had been previously coated with collagen and cultured for 24 h [3, 4]. Contractile activity was induced by electrical stimulation [3, 4]. Fibres were loaded with 4-Amino-5-Methylamino-2’,7’-Difluorofluorescein Diacetate (DAF-FM DA), Dihydroethidium (DHE) or 2’,7’-Dichlorofluorescein Diacetate (DCFH DA), fluorescent probes for the assessment of Nitric Oxide (NO^.), Superoxide (O₂−^.) and a general probe for ROS respectively. Unpaired Student’s t test for single comparisons, significance p<0.05. Results: At rest the nNOS mice had a higher rate of increase in DAF-FM fluorescence compared with the control group implying a higher NO^. generation (p<0.05). However, no significant differences were observed between the stimulated fibres from the two groups. Ethidium fluorescence from DHE-loaded fibres increased in both groups indicating a potential increase in the rate of O₂−^. generation in the sarcoplasm after contractions (p<0.05) with no difference between the rates in wild type and transgenic mice. Fibres from nNOS overexpressors showed a decrease in ethidium fluorescence compared with the fibres from control mice at rest (p<0.05). Following DCFH loading, no differences were seen in DCF fluorescence between the groups. Discussion: Contractile activity increased ROS and RNS in fibres from both wild type and nNOS overexpressing mice. The mice overexpressing nNOS appeared to generate more NO^. at rest compared with those from control mice. However following contractile activity the fluorescence from fibres did not differ between the groups. These data indicate that overexpression of nNOS can increase NO^. bioavailability in skeletal muscle fibres and reduce the availability of O₂−^..