Low-frequency fatigue is a form of muscle fatigue that follows intense muscle activity and is characterized by reduced tetanic tension at low frequencies of stimulation while tetanic tension at high stimulus frequencies is close to normal. The present experiments were performed on isolated single fibers of mouse in which tension and intracellular calcium concentration ([Ca2+]i) were measured. Fatigue was produced by intermittent short tetani continued until tension had declined to 30% of control. Comparison of low- (30- and 50-Hz) and high- (100-Hz) frequency tetani under control conditions and after 30 min of recovery from fatigue showed that low-frequency fatigue was present. During low-frequency fatigue, tetanic [Ca2+]i was substantially reduced at all stimulus frequencies but there was no change in Ca2+ sensitivity or maximum Ca(2+)-activated tension. One possible cause of the reduced tetanic [Ca2+]i is failure of conduction of the action potential in the T tubule, leading to reduced [Ca2+]i in the center of the fiber. However, imaging of [Ca2+]i across the fiber during low-frequency fatigue did not show any such gradient, suggesting that Ca2+ release is uniform across the fiber. Another possible mechanism is that changes in the Ca2+ pumping ability of the sarcoplasmic reticulum might affect tetanic [Ca2+]i. Measurements of the sarcoplasmic reticulum pump function showed a small slowing of Ca2+ uptake rate during low-frequency fatigue, which is unlikely to cause the reduced tetanic [Ca2+]i. In conclusion, the immediate cause of low-frequency fatigue appears to be a reduced tetanic [Ca2+]i, which is probably a consequence of a reduced Ca2+ release from the sarcoplasmic reticulum.