In skeletal muscle, the release of calcium (Ca²⁺) by ryanodine sensitive sarcoplasmic reticulum (SR) Ca²⁺ release channels (i.e., ryanodine receptors; RyR1s) is the primary determinant of contractile filament activation. Much attention has been focused on calsequestrin (CASQ1) and its role in SR Ca²⁺ buffering as well as its potential for modulating RyR1, the L-type Ca²⁺ channel (dihydropyridine receptor, DHPR) and other sarcolemmal channels through sensing luminal [Ca²⁺]. The genetic ablation of CASQ1 expression results in significant alterations in SR Ca²⁺ content and SR Ca²⁺ release especially during prolonged activation. While these findings predict a significant loss-of-function phenotype in vivo, little information on functional status of CASQ1 null mice is available. We examined fast muscle in vivo and in vitro and identified significant deficits in functional performance that indicate an inability to sustain contractile activation. In single CASQ1 null skeletal myofibers we demonstrate a decrease in voltage dependent RyR Ca²⁺ release with single action potentials and a collapse of the Ca²⁺ release with repetitive trains. Under voltage clamp, SR Ca²⁺ release flux and total SR Ca²⁺ release are significantly reduced in CASQ1 null myofibers. The decrease in peak Ca²⁺ release flux appears to be solely due to elimination of the slowly decaying component of SR Ca²⁺ release, whereas the rapidly decaying component of SR Ca²⁺ release is not altered in either amplitude or time course in CASQ1 null fibers. Finally, intra-SR [Ca²⁺] during ligand and voltage activation of RyR1 revealed a significant decrease in the SR[Ca²⁺]_free in intact CASQ1 null fibers and a increase in the release and uptake kinetics consistent with a depletion of intra-SR Ca²⁺ buffering capacity. Taken together we have revealed that the genetic ablation of CASQ1 expression results in significant functional deficits consistent with a decrease in the slowly decaying component of SR Ca²⁺ release.