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+](o) in 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+](o) or 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+](o) protects against fatigue rather than inducing it and, second, that a considerable depletion of [Ca2+](o) in the transverse tubules may contribute to fatigue.