Fluorescence microscopy has been used to examine the distribution of intracellular calcium concentration ([Ca2+](i)) in isolated single fibres from the mouse flexor brevis muscle during fatiguing stimulation. Under control conditions there was a virtually uniform distribution of [Ca2+](i) in fura-2 loaded fibres either at rest or during short (0.35 s, 100 Hz) tetani. Fatigue produced by repeated short tetani was accompanied by an early rise, followed by a marked fall, in tetanic [Ca2+](i). Throughout the period of fatiguing stimulation the distribution of [Ca2+](i) remained uniform with no detectable gradients observed. In contrast, when fatigue was produced by continuous 100 Hz stimulation, a small gradient of [Ca2+](i) developed across the fibre with the [Ca2+](i) in the centre of the fibre lower than that at the edge of the fibre. This gradient was apparent after 1.7 s, persisted for at least 11 s and was superimposed on a rise followed by a fall in spatially averaged [Ca2+](i). Reduction of the extracellular Na+ to 50% caused reduced force production and a reduced [Ca2+](i) in the centre of the fibre. To assess the contribution of reduced response of the myofibrillar proteins to [Ca2+](i) during continuous tetani, the relation between [Ca2+](i) and force throughout the long tetanus was compared with that obtained in short, unfatigued tetani. These results show that in long tetani, reduced tetanic [Ca2+](i) and reduced responsiveness of the myofibrillar proteins to [Ca2+](i) each make important contributions to the decline of force, whereas the gradients of [Ca2+](i) make only a small contribution.
