Intra-sarcomeric gradients of [Ca2+] during activation of action potential stimulated frog single fibres were investigated with the Ca2+ indicator fluo-3 and confocal and two-photon microscopy. The object of these experiments was to look for evidence of extra-junctional Ca2+ release and examine the microscopic diffusion of Ca2+ within the sarcomere. By exploiting the spatial periodicity of sarcomeres within the fibre, we could achieve a high effective line-scanning rate ( approximately 8000 lines s-1), although the laser scanning microscope was limited to < 1000 lines s-1. At this high time resolution, the time course of fluorescence changes was very different at the z- and m-lines, with a significant delay ( approximately 1 ms; 22 C) between the rise of fluorescence at the z-line and the m-line. To calculate the expected fluorescence changes, we used a multi-compartment model of Ca2+ movements in the half-sarcomere in which Ca2+ release was restricted to triadic junctions (located at z-lines). Optical blurring by the microscope was simulated to generate fluorescence signals which could be compared directly to experimental data. The model which reproduced our experimental findings most accurately included Ca2+ binding by ATP, as well as indicator binding to immobile sarcomeric proteins. After taking sarcomeric misregistration within the fibre into account, there was very good agreement between the model and experimental results. We conclude that there is no experimental evidence for Ca2+ release at locations other than at z-lines. In addition, our calculations support the conclusion that rapidly diffusing Ca2+ buffers (such as ATP) are important in shaping the Ca2+ transient and that the details of intracellular indicator binding need to be considered to explain correctly the time course of fluorescence change in the fibre.