A possible role of extracellular Cl(-) concentration ([Cl(-)](o)) in fatigue was investigated in isolated skeletal muscles of the mouse. When [Cl(-)](o) was lowered from 128 to 10 mM, peak tetanic force was unchanged, fade was exacerbated (wire stimulation electrodes), and a hump appeared during tetanic relaxation in both nonfatigued slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles. Low [Cl(-)](o) increased the rate of fatigue 1) with prolonged, continuous tetanic stimulation in soleus, 2) with repeated intermittent tetanic stimulation in soleus or EDL, and 3) to a greater extent with repeated tetanic stimulation when wire stimulation electrodes were used rather than plate stimulation electrodes in soleus. In nonfatigued soleus muscles, application of 9 mM K(+) with low [Cl(-)](o) caused more rapid and greater tetanic force depression, along with greater depolarization, than was evident at normal [Cl(-)](o). These effects of raised [K(+)](o) and low [Cl(-)](o) were synergistic. From these data, we suggest that normal [Cl(-)](o) provides protection against fatigue involving high-intensity contractions in both fast- and slow-twitch mammalian muscle. This phenomenon possibly involves attenuation of the depolarization caused by stimulation- or exercise-induced run-down of the transsarcolemmal K(+) gradient.