Periods of low frequency stimulation are known to increase the net Ca(2+) uptake in skeletal muscle but the mechanism responsible for this Ca(2+) entry is not known. In this study a novel high-resolution fluorescence microscopy approach allowed the detection of an action potential-induced Ca(2+) flux across the tubular (t-) system of rat extensor digitorum longus muscle fibres that appears to be responsible for the net uptake of Ca(2+) in working muscle. Action potentials were triggered in the t-system of mechanically skinned fibres from rat by brief field stimulation and t-system [Ca(2+)] ([Ca(2+)](t-sys)) and cytoplasmic [Ca(2+)] ([Ca(2+)](cyto)) were simultaneously resolved on a confocal microscope. When initial [Ca(2+)](t-sys) was > or = 0.2 mM a Ca(2+) flux from t-system to the cytoplasm was observed following a single action potential. The action potential-induced Ca(2+) flux and associated t-system Ca(2+) permeability decayed exponentially and displayed inactivation characteristics such that further Ca(2+) entry across the t-system could not be observed after 2-3 action potentials at 10 Hz stimulation rate. When [Ca(2+)](t-sys) was closer to 0.1 mM, a transient rise in [Ca(2+)](t-sys) was observed almost concurrently with the increase in [Ca(2+)](cyto) following the action potential. The change in direction of Ca(2+) flux was consistent with changes in the direction of the driving force for Ca(2+). This is the first demonstration of a rapid t-system Ca(2+) flux associated with a single action potential in mammalian skeletal muscle. The properties of this channel are inconsistent with a flux through the L-type Ca(2+) channel suggesting that an as yet unidentified t-system protein is conducting this current. This action potential-activated Ca(2+) flux provides an explanation for the previously described Ca(2+) entry and accumulation observed with prolonged, intermittent muscle activity.