JP-45, a 45 kDa transmembrane protein of skeletal muscle sarcoplasmic reticulum, interacts with the alfa1.1 subunit of the dihydropyridine receptor (Cav1.1) and calsequestrin (CASQ), two key proteins of the EC-coupling machinery. The JP-45 protein may be a component of a signaling pathway connecting the SR lumen (CASQ) to the Cav1.1. We generated JP-45 and CASQ1 double knockout (dKO) mice and compared their phenotype with age-matched wild-type (WT) littermates. Analysis of spontaneous motor activity revealed no difference in the running distance between dKO and WT mice. The intracellular peak Ca2+ signal evoked by a 0.5 ms pulse at supramaximal field stimulation was lower in FDB fibers from JP-45 null, CASQ1 null and dKO mice compared to WT. The global Ca2+ signal evoked by a train of pulses at 100 Hz for 300ms was negligibly affected by La3+ in WT, JP-45 null and CASQ1 null fibers, but strongly inhibited in fibers from dKO mice. Mn2+ quenching experiments in fura-2 loaded FDB fibers indicate that Ca2+ influx accounts for most of the myoplasmic [Ca2+]i increase during tetanic stimulation in fibers from dKO mice. The Mn2+ influx was blocked by 50 μM nifedipine an inhibitor of Ca2+ currents via Cav1.1. These results demonstrate that lack of JP-45 and CASQ-1 upregulate Ca2+ influx via Cav1.1.