The cytosolic free Ca2+-concentrations transients elicited during muscular contraction are well characterized but little is known about the free [Ca2+] dynamics inside the sarcoplasmic reticulum (SR). FRET-based Ca2+ indicators (Cameleons) allowed us to investigate SR Ca2+-handling with high time resolution (9 ms or less). The impact of calsequestrin (CS) on SR [Ca2+] was studied in enzymatically dissociated FDB muscle fibers from wild type (WT), CS type 1(-/-) (KO) and double CS type 1/CS type 2(-/-) (DKO) mice 7 days after transfection with the cDNA coding for D1ER.[Ca2+] measurements were performed at rest and during repetitive stimulation at 1, 5, 20 and 60 Hz, and the YFP(535nm)/CFP(480nm) ratio was deemed as a measure of the free SR [Ca2+].The SR [Ca2+] at rest did not differ between WT (n=26), KO (n=25) and DKO (n=21) fibers while during electrical stimulation they were rather small in WT, reflecting powerful buffering of SR [Ca2+]. In KO and DKO fibers, a significant reduction in the SR [Ca2+] occurred, which increased in parallel with the stimulation frequency. At 60 Hz the SR became virtually depleted of Ca2+, both in KO and DKO fibers. Calcium reuptake during and after the trains of stimuli was governed by 3 rate constants of 50 s−1, 1-5 s−1 and 0.3 s−1 (at 26°C). In conclusion, CS-KO fibers represent a unique model to resolve the kinetics of SR release and reuptake.