Single mechanically skinned extensor digitorum Longus (EDL) rat fibres were used as a model to study the influence of functional t-tubules on the properties of RyR1 in adult skeletal muscle. Fibres were superfused with solutions approximating to the intracellular milieu. Following skinning, the t-tubules re-seal and repolarise, allowing the sarcoplasmic reticulum (SR) Ca2+ release to be activated by field stimulation. However, in the present study, some fibres exhibited localised regions where depolarisation-induced SR Ca2+ release was absent, due to failure of the t-tubules to re-seal. When these fibres were exposed to caffeine to directly activate RyR1, regions with re-sealed t-tubules exhibited greater sensitivity to submaximal (2-5 mM) levels of caffeine (n = 8), while the response to a supramaximal SR Ca2+ release stimulus was uniform (n = 8, p < 0.05). This difference in RyR1 sensitivity was unaffected by sustained depolarisation of the t-tubule network. However, after saponin permeabilization of the t-tubules or withdrawal of Ca2+ from the t-tubules before skinning, the difference in agonist sensitivity was abolished. These results suggest that in adult skeletal muscle fibres, the presence of a functional t-tubule network increases the sensitivity of RyR1 to agonists via a mechanism that involves binding of Ca2+ to an extracellular regulatory site.