Calsequestrin (CSQ) is a high capacity Ca(2+)-binding protein present in the lumen of sarcoplasmic reticulum (SR) in striated muscle cells and has been shown to regulate the ryanodine receptor Ca(2+) release channel activity through interaction with other proteins present in the SR. Here we show that overexpression of wild-type CSQ or a CSQ mutant lacking the junction binding region (amino acids 86-191; Delta junc-CSQ) in mouse skeletal C2C12 myotube enhanced caffeine- and voltage-induced Ca(2+) release by increasing the Ca(2+) load in SR, whereas overexpression of a mutant CSQ lacking a Ca(2+) binding, aspartate-rich domain (amino acids 352-367; Delta asp-CSQ) showed the opposite effects. Depletion of SR Ca(2+) by thapsigargin initiated store-operated Ca(2+) entry (SOCE) in C2C12 myotubes. A large component of SOCE was inhibited by overexpression of wild-type CSQ or Delta junc-CSQ, whereas myotubes transfected with Delta asp-CSQ exhibited normal function of SOCE. These results indicate that the aspartate-rich segment of CSQ, under conditions of overexpression, can sustain structural interactions that interfere with the SOCE mechanism. Such retrograde activation mechanisms are possibly taking place at the junctional site of the SR.