1. The effects of wortmannin and 2-(4-morpholinyl)-8-phenyl-1[4H]-benzopyran-4-one (LY294002), inhibitors of phosphatidylinositol 3-kinase, on the contractile responses of murine skeletal muscle were studied. Wortmannin (10-100 microM) suppressed twitch and tetanic contraction evoked by field stimulation of diaphragm without causing elevation of muscle tone. The inhibition was quasi-irreversible with IC50 approximately 15 microM. In contrast, LY294002 increased twitch responses and elevated muscle tone. 2. Wortmannin reversibly depressed the maximal slope of action potential upstroke by approximately 40% and inhibited the membrane depolarization and spontaneous burst of action potential induced by crotamine, a polypeptide toxin that activates the Na+ channel of skeletal muscle. 3. Wortmannin inhibited contractures evoked by high K+, ryanodine and caffeine, but potentiated the contracture induced by rapamycin, which binds to myoplasmic FK506 binding protein, an immunophilin closely associated with the ryanodine receptor. The contractures elicited by cardiotoxin, which disrupts the integrity of sarcolemma and thereby elevates 'myoplasmic' Ca2+ level, were suppressed only slightly. 4. In placed left atrium and ventricular strip, wortmannin and LY294002 produced a positive inotropic effect. 5. The results suggest that, in addition to depressing the Ca2+ mobilization from sarcoplasmic reticulum, wortmannin exerts a novel inhibitory action on the excitation-contraction coupling in skeletal muscle but not in cardiac muscle.