Isolated Rana temporaria sartorius muscle fibres were subject to introduction and subsequent withdrawal of 400 mM extracellular glycerol, exposures to high divalent ion concentrations and then cooling. Tubular detachment was then assessed through changes in the action potential afterdepolarization. (1) The rapid (5-10 min) rather than slow cooling step (30 min) produced a gradual (30 min) development of detubulation arrested by the subsequent replacement of glycerol and reversed by addition of 350 mM sucrose. Such osmotic agents influenced neither resting potentials of intact or detubulated fibres nor action potentials in intact fibres. (2) Full tubular detachment was achieved by 40 min. Laser epifluorescence microscopy demonstrated an accompanying tubular vacuolation through its trapping of a Rhodamine dye. (3) Subsequent re-additions (at 10-80 min) of glycerol restored the afterdepolarization in 30% of detubulated fibres and correspondingly reduced vacuolation. Sustained (> 60 min) exposures to 350 mM sucrose, applied between 30-60 min, both reversed tubular isolation in 70% of detubulated fibres and abolished tubular vacuolation. Finally, results from transient (10-30 min) sucrose exposures resembled the consequences of sustained applications of glycerol, suggesting that detubulation and its reversal result from an osmotic mechanism. (4) Nevertheless, irreversible changes developed after 70-80 min in 70% of detubulated fibres, a process hastened by slow cooling steps in the initial osmotic stress. The present study thus correlates morphological and electrophysiological consequences of applying osmotic shock to skeletal muscle for the first time. It additionally differentiates reversible and irreversible components of detubulation. Finally, it suggests that detubulation results from the similarly reversible vacuolation observed under comparable osmotic conditions, and that such vacuolation can eventually lead to irreversible detubulation.