The contribution of metabolic energy to the degradation of intracellular proteins in skeletal muscle was investigated. Isolated chick skeletal muscles deprived of oxygen and muscles incubated in buffer under nonphysiological conditions containing inhibitors of glycolysis and mitochondrial respiration had lower concentrations or undetectable levels of ATP and faster rates of proteolysis. Both total protein breakdown and the breakdown of myofibrillar proteins were stimulated 35-124% in ATP-depleted tissues. However, ATP-depleted muscles incubated in buffer to which no Ca2+ was added showed slower rates of total protein breakdown and no significant change in myofibrillar proteolysis compared with control muscles. Trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64), a compound that inhibits the calpains and the lysosomal cysteine proteases, completely blocked the Ca(2+)-stimulated breakdown of nonmyofibrillar and myofibrillar proteins in ATP-depleted muscles. However, Ca(2+)-stimulated proteolysis was not inhibited in ATP-depleted muscles incubated with weak bases to prevent lysosome function. These data suggest that intracellular proteins can be degraded in skeletal muscle in the absence of metabolic energy and that the calpains play a major role in the enhanced proteolysis in skeletal muscles depleted of ATP.