Confocal imaging of impermeant fluorescent dyes trapped in the tubular (t‐) system of skeletal muscle fibres of rat and cane toad was used to examine changes in the morphology of the t‐system upon mechanical skinning, the time course of dye loss from the sealed t‐system in mechanically skinned fibres and the influence of rapid application and removal of glycerol on the morphology of the sealed t‐system. In contrast to intact fibres, which have a t‐system open to the outside, the sealed t‐system of toad mechanically skinned fibres consistently displayed local swellings (vesicles). The occurrence of vesicles in the sealed t‐system of rat‐skinned fibres was infrequent. Application and removal of 200–400mM glycerol to the sealed t‐system did not produce any obvious changes in its morphology. The dyes fluo‐3, fura‐2 and Oregon green 488 were lost from the sealed t‐system of toad fibres at different rates suggesting that the mechanism of organic anion transport across the tubular wall was not by indiscriminate bulk transport. The rate of fluo‐3 and fura‐2 loss from the sealed t‐system of rat fibres was greater in rat than in toad fibres and could be explained by differences in surface area: volume ratio of the t‐system in the two fibre types. Based on the results presented here and on other results from this laboratory, an explanation is given for the formation of numerous vesicles in toad‐skinned fibres and lack of vesicle formation in rat‐skinned fibres. This explanation can also help with better understanding the mechanism responsible for vacuole formation in intact fibres.