The possible role of altered extracellular Ca²⁺concentration ([Ca²⁺]_o) in skeletal muscle fatigue was tested on isolated slow-twitch soleus and fast-twitch extensor digitorum longus muscles of the mouse. The following findings were made. 1) A change from the control solution (1.3 mM [Ca²⁺]_o) to 10 mM [Ca²⁺]_o, or to nominally Ca²⁺-free solutions, had little effect on tetanic force in nonfatigued muscle.2) Almost complete restoration of tetanic force was induced by 10 mM [Ca²⁺]_oin severely K⁺-depressed muscle (extracellular K⁺ concentration of 10–12 mM). This effect was attributed to a 5-mV reversal of the K⁺-induced depolarization and subsequent restoration of ability to generate action potentials (inferred by using the twitch force-stimulation strength relationship).3) Tetanic force depressed by lowered extracellular Na⁺concentration (40 mM) was further reduced with 10 mM [Ca²⁺]_o.4) Tetanic force loss at elevated extracellular K⁺ concentration (8 mM) and lowered extracellular Na⁺concentration (100 mM) was partially reversed with 10 mM [Ca²⁺]_oor markedly exacerbated with low [Ca²⁺]_o.5) Fatigue induced by using repeated tetani in soleus was attenuated at 10 mM [Ca²⁺]_o(due to increased resting and evoked forces) and exacerbated at low [Ca²⁺]_o. These combined results suggest, first, that raised [Ca²⁺]_oprotects against fatigue rather than inducing it and, second, that a considerable depletion of [Ca²⁺]_oin the transverse tubules may contribute to fatigue.