The main objective of this study was to analyse glycogen in single muscle fibres, using a recently developed microfluorometric method which detects subpicomol amounts of NADPH, glucose and glycogen (as glucosyl units) (detection limit 0.16-0.17 pmol in a 25 nl sample) without fluorochrome amplification. The fibres were freshly dissected from the twitch region of the iliofibularis muscle of the cane toad (Bufo marinus), and were mechanically skinned under paraffin oil to gain access to the intracellular compartments. The results show that (1) glycogen concentrations in toad skeletal muscle fibres range between 25.8 and 369 mmol glucosyl units/litre fibre volume; (2) there is a large variation in glycogen content between individual fibres from the iliofibularis muscle of one animal; (3) there are seasonal differences in the glycogen content of toad single muscle fibres; (4) the total amount of glycogen in single muscle fibres of the toad does not decrease significantly when storing the tissue, under paraffin oil, at 20-25 degree C for up to 6 h or at 4 degree C for up to 24 h; and (5) 15-26% of fibre glycogen can be washed in an aqueous solution at pH 5-7, within 5 min, while 74-85% of fibre glycogen remains associated with the washed skinned fibre, even after 40 min exposure of the skinned fibre preparation to the aqueous environment. The retention of most glycogen in the fibre preparation after mechanical removal of the plasma membrane and extensive washing indicates that in toad skeletal muscle fibres the largest proportion of glycogen is tightly bound to intracellular structures. The results also show that the skinned muscle fibre preparation is well suited for microfluorometric glycogen determination, since low molecular weight non-glycogen contributors to the fluorescence signal can be removed from the myoplasmic space prior to the glycogen hydrolysis step.