The tubular (t) system is essential for normal function of skeletal muscle fibre, acting as a conduit for molecules and ions within the cell. However, t system accessibility and interconnectivity have been mainly assessed in fixed cells where the t system no longer fully represents that of the living cell. Here, fluorescent dyes of different diameter were allowed to equilibrate within the t system of intact fibres from toad, mechanically skinned to trap the dyes, and then imaged using confocal microscopy to investigate t system accessibility and interconnectivity. Dual imaging of rhod-2 and a 500 kDa fluorescein dextran identified regions throughout the t system that differed in the accessibility to molecules of different molecular weight. Restrictions within the t system lumen occurred at the junctions of the longitudinal and transverse tubules and also where a transverse tubule split into two tubules to maintain their alignment with Z-lines of adjacent mis-registered sarcomeres. Thus, three types of tubule, transverse, longitudinal and Z, can be identified by their lumenal diameter in this network. The latter we define for the first time as a tubule with a narrow lumen that is responsible for the change in register. Stretch-induced t system vacuolation showed exclusive access of rhod-2 to these structures indicating their origin was the longitudinal tubules. Exposing the sealed t system to highly hypertonic solution reversed vacuolation of longitudinal tubules and also revealed that these tubules are not collapsible. Fluorescence recovery after photobleaching (FRAP) measurements of t system-trapped fluo-5 N showed interconnectivity through the t system along the axis of the fibre. However, diffusion occurred at a rate slower than expected given the known number of longitudinal tubules linking adjacent transverse tubules. This could be explained by the observed narrow opening to the longitudinal tubules from transverse tubules, reducing the effective cross-sectional area in which molecules could move within the t system.