The volume of the extracellular compartment (tubular system) within intact muscle fibres from cane toad and rat was measured under various conditions using confocal microscopy. Under physiological conditions at rest, the fractional volume of the tubular system (t-sys(Vol)) was 1.38 +/- 0.09 % (n = 17), 1.41 +/- 0.09 % (n = 12) and 0.83 +/- 0.07 % (n = 12) of the total fibre volume in the twitch fibres from toad iliofibularis muscle, rat extensor digitorum longus muscle and rat soleus muscle, respectively. In toad muscle fibres, the t-sys(Vol) decreased by 30 % when the tubular system was fully depolarized and decreased by 15 % when membrane cholesterol was depleted from the tubular system with methyl-beta-cyclodextrin but did not change as the sarcomere length was changed from 1.93 to 3.30 microm. There was also an increase by 30 % and a decrease by 25 % in t-sys(Vol) when toad fibres were equilibrated in solutions that were 2.5-fold hypertonic and 50 % hypotonic, respectively. When the changes in total fibre volume were taken into consideration, the t-sys(Vol) expressed as a percentage of the isotonic fibre volume did actually decrease as tonicity increased, revealing that the tubular system in intact fibres cannot be compressed below 0.9 % of the isotonic fibre volume. The results can be explained in terms of forces acting at the level of the tubular wall. These observations have important physiological implications showing that the tubular system is a dynamic membrane structure capable of changing its volume in response to the membrane potential, cholesterol depletion and osmotic stress but not when the sarcomere length is changed in resting muscle.