The intracellular Ca(2+) concentration ([Ca(2+)](i)) in skeletal muscles must be rapidly regulated during the excitation-contraction-relaxation process. However, the signalling components involved in such rapid Ca(2+) movement are not fully understood. Here we report that mice deficient in the newly identified PtdInsP (phosphatidylinositol phosphate) phosphatase MIP/MTMR14 (muscle-specific inositol phosphatase) show muscle weakness and fatigue. Muscles isolated from MIP/MTMR14(-/-) mice produced less contractile force, had markedly prolonged relaxation and showed exacerbated fatigue relative to normal muscles. Further analyses revealed that MIP/MTMR14 deficiency resulted in spontaneous Ca(2+) leakage from the internal store - the sarcoplasmic reticulum. This was attributed to decreased metabolism (dephosphorylation) and the subsequent accumulation of MIP/MTMR14 substrates, especially PtdIns(3,5)P(2) and PtdIns (3,4)P(2). Furthermore, we found that PtdIns(3,5)P(2) and PtdIns(3,4)P(2) bound to, and directly activated, the Ca(2+) release channel (ryanodine receptor 1, RyR1) of the sarcoplasmic reticulum. These studies provide the first evidence that finely controlled PtdInsP levels in muscle cells are essential for maintaining Ca(2+) homeostasis and muscle performance.