Nitric oxide (NO) is thought to play multiple roles in skeletal muscle including regulation of some adaptations to contractile activity, but appropriate methods for the analysis of intracellular NO activity are lacking. In this study we have examined the intracellular generation of NO in isolated single mature mouse skeletal muscle fibres at rest and following a period of contractile activity. Muscle fibres were isolated from the flexor digitorum brevis muscle of mice and intracellular NO production was visualized in real-time using the fluorescent NO probe 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA). Some leakage of DAF-FM was apparent from fibres loaded with the probe, but they retained sufficient probe to respond to changes in intracellular NO following addition of the NO donor 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) up to 30 min after loading. Electrically stimulated contractions in isolated fibres increased the rate of change in DAF-FM fluorescence by approximately 48% compared to non-stimulated fibres (P < 0.05) and the rate of change in DAF-FM fluorescence in the stimulated fibres returned to control values by 5 min after contractions. Treatment of isolated fibres with the NO synthase inhibitors NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) or NG-monomethyl-L-arginine (L-NMMA) reduced the increase in DAF-FM fluorescence observed in response to contractions of untreated fibres. Treatment of fibres with the cell-permeable superoxide scavenger 4,5-dihydroxy-1,3-benzenedisulphonic acid (Tiron) also reduced the increase in fluorescence observed during contractions suggesting that superoxide, or more probably peroxynitrite, contributes to the fluorescence observed. Thus this technique can be used to examine NO generation in quiescent and contracting skeletal muscle fibres in real time, although peroxynitrite and other reactive nitrogen species may potentially contribute to the fluorescence values observed.