The tubular (t-) system of skeletal muscle is an internalization of the plasma membrane. The t-system forms a junction with the terminal cisternae of the sarcoplasmic reticulum (SR) at every sarcomere of skeletal muscle. At any given moment the [Ca2+] within the small volume bound by the junctional membranes will be critically determined by the leak of Ca2+ through ryanodine receptors and the net Ca2+ handling ability of the t-system. To assess the Ca2+ uptake and handling ability of the t-system we trapped fluo-5N or rhod-5N in the t-system of mechanically skinned fibres of rat extensor digitorum longus muscle and continuously imaged t-system Ca2+-dependent fluorescence on a confocal microscope. In situ calibration determined the half signal of fluo-5N and rhod-5N to be 335 and 872 µM, respectively. Rhod-5N was selected for ongoing experiments. Chronic depletion of [Ca2+]SR with caffeine reduced [Ca2+]t-sys to 0.1 mM via chronic activation of store-operated Ca2+ entry (SOCE). We then exposed Ca2+-depleted preparations to 0-800 nM [Ca2+]cyto in 50 mM EGTA. At [Ca2+]cyto > 100 nM the [Ca2+]t-sys reached a plateau at 1.8-1.9 mM after 3-5 s. At [Ca2+]cyto < 100 nM the [Ca2+]t-sys did not always reach this plateau and showed a biphasic uptake of Ca2+. At the plateau [Ca2+]t-sys lowering [Ca2+]cyto to < 1 nM did not cause a significant loss of [Ca2+]t-sys. There was an apparent absence of effect of removing [Na+]cyto on these results. Ca2+ fluxes across the t-system during uptake and SOCE were determined by multiplying the time derivative of [Ca2+]t-sys by buffering capacity and t-system SA:V. Mathematical modeling of these results suggests that the plasma membrane CaATPase (PMCA) with its low Km for Ca2+ is the major protein responsible for t-system Ca2+ uptake in the resting muscle, despite the higher transport capacity of the Na-Ca exchanger.