Experiments were carried out to investigate excitation-contraction coupling in vertebrate skeletal muscle fibers. Single-dissected twitch fibers of frog muscle were injected with flourescent calcium (Ca) indicators, and fluorescence intensity (F) and changes in intensity (ΔF) were recorded during various stimulation protocols (16-22 °C). In fibers activated by an action potential, the low-affinity indicator furaptra was used to estimate the spatially-averaged change in myoplasmic free calcium concentration (ΔC[a]; peak value, 15-20 μM; time to peak, 4-5 ms). Δ[Ca], in combination with a kinetic model, was used to calculate the change in concentration of calcium bound to the principal myoplasmic Ca buffers (ATP, troponin, parvalbumin). The rate of Ca release from the sarcoplasmic reticulum (SR) was obtained from the time derivative of the estimated change in total myoplasmic calcium concentration, Δ[CaT] (= Δ[Ca] + Δ[CaATP] + Δ[CaTroponin] + Δ[CaParvalbumin]).