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Since there are uncertainties about the concentration of free cytosolic Ca2+ reached in a muscle fiber during a transient elicited by electric stimulation, and about the KdCa2+ of the fast dye Magfluo-4, we performed two groups of experiments aimed at calibrating the Ca2+ transients obtained with this dye. For the fiber experiments, we used murine single skeletal muscle fibers obtained from enzymatically dissociated flexor digitorum brevis, loaded with 8-10 μM of the dye in its acetoxymethyl ester form, during 40 min at 21-22°C, to determine Fmax, Fmin and dye compartmentalization. The cells were permeabilized with saponine and exposed to 50 mM of free calcium to intracellularly saturate the dye. Compartmentalization was assessed using digitonin and triton-X100. For our conditions, Fmax and Fmin were 160±14 (n=9, mean±SEM) and 3.4±0.7 (n=6). Dye compartmentalization was found to be less than 10% (n=1). For the in vitro experiments, KdCa2+ (20°C) values of 21.6 μM, 27.8 μM and 29.1 μM were obtained in presence of 0, 1 and 2 mM free Mg2+, respectively. To calculate Ca2+ concentration from the fluorescence signals, we used the methodologies proposed by Grynkiewicz et al. (1985) and by Caputo et al. (1994). Preliminary results, taking into account our data or some values from the literature (Baylor et al. 2009), show single Ca2+ transient's peak between 2 and 7 μM (n=4). We are now calculating the kinetics of Ca2+ transients and the quantitative changes induced by modulating Ca2+ release and sequestration mechanisms in different fiber types. In conclusion, we have calculated the peak Ca2+ concentration after single stimulation in fibers loaded with Magfluo-4. Our preliminary results are comparable to those obtained with other fast Ca2+ dyes, such as Magfura-5, but lower than those reported with Magfura-2.