Intracellular inorganic phosphate increases during muscle fatigue and may be responsible for certain of the changes in muscle function observed in fatigue. To test this hypothesis inorganic phosphate was micro-injected in single mouse muscle fibres which were also injected with indo-1 to measure intracellular Ca²⁺. Following phosphate injection, intracellular Ca²⁺, both at rest and during tetani, was reduced as was tetanic force. The rate at which the sarcoplasmic reticulum (SR) pumped Ca²⁺ out of the myoplasm was accelerated following phosphate injection. Intracellular Ca²⁺ and force recovered over 1-h. The changes in maximum Ca²⁺-activated force and Ca²⁺ sensitivity which would be expected if the phosphate remained in the myoplasm were largely absent. The most likely interpretation is that inorganic phosphate enters the SR where it precipitates with Ca²⁺ and thereby reduced release of Ca²⁺ from the SR and accelerated the rate of uptake of Ca²⁺ by the pump. The 1-h recovery may represent the entry of additional Ca²⁺ into the cell to reestablish the normal gradient of Ca²⁺ across the sarcolemma.