Previous work on cultured myotubes provided evidence that the presence of functional t-tubules is associated with a decrease in the open probability of associated ryanodine receptors (RyRs), located in the junctional SR (Zhou et al. 2006). It was proposed that the underlying mechanism might involve a tonic inhibitory influence of dihydropyridine receptors (DHPRs) on coupled RyRs. We have used mechanically skinned fibres as a model to study the influence of functional t-tubules on RyR1 in adult skeletal muscle. Rats (200-250 g) were humanely killed. Single extensor digitorium longus (EDL) fibres were mechanically skinned under oil and then perfused with solutions approximating to the intracellular milieu. The perfusing solution contained fluo-3 acid (10 μM) to allow detection of cytosolic Ca²⁺ using confocal microscopy. Following skinning, the t-tubules can reseal and repolarise, allowing the physiological SR Ca²⁺ release mechanism to be triggered by field stimulation. In approximately 25% of fibres, field stimulation resulted in non-uniform (“patchy”) Ca²⁺ release. Experiments involving trapping of fluo-5N in the t-tubules revealed that the non-responding areas corresponded to regions in which the t-tubules had failed to re-seal. Fibres exhibiting patchy Ca²⁺ release were exposed to sequentially to 5 and then 20 mM caffeine. Under these conditions, it was found that 5 mM caffeine only induced Ca²⁺ release in regions of each fibre with re-sealed t-tubules (n=8). However, subsequent exposure to 20 mM caffeine induced Ca²⁺ release in both sealed and un-sealed areas (n=8). Following Ca²⁺reloading of the SR, and readdition of 20 mM caffeine, there was no significant difference between the Ca²⁺ transient amplitude in the sealed and unsealed regions, suggesting a similar SR Ca²⁺ content (n=8, p<0.05). The higher sensitivity to caffeine was unaffected by depolarisation of the re-sealed t-tubule network following substitution of K⁺ with Na⁺ in the perfusing solution. However, fibres washed with Ca²⁺ -free (1 mM EGTA) Tyrode’s solution, prior to skinning under oil, did not exhibit differential sensitivity to caffeine in areas with sealed and un-sealed t-tubules. These data suggest that in adult EDL muscle fibres the presence of functional re-sealed t-tubules increases the sensitivity of RyR1 to activation by caffeine. This effect is apparently not dependent upon polarisation of the membrane per se, but rather the presence of Ca²⁺ in the re-sealed t-tubules. It is possible that Ca²⁺ may influence RyR1 sensitivity (i) by influencing the conformational state of the DHPR or (ii) via a localised efflux from the re-sealed t-tubule.