1. The effect of low [ATP] on depolarization-induced Ca2+ release from the sarcoplasmic reticulum (SR) was examined using mechanically skinned skeletal muscle fibres of the toad. The amount of Ca2+ released was determined by examining the extent of SR Ca2+ depletion following a single depolarization in the presence of specified total [ATP] and free [Mg2+] and 1 mM EGTA (< 10 nM Ca2+). 2. Reducing the total [ATP] from 8 to 2 mM did not significantly affect the extent of depolarization-induced Ca2+ release. When the total [ATP] was lowered to 0.5 mM, depolarization-induced Ca2+ release was markedly reduced. The additional presence of 1.5 mM AMP did not reverse this effect of low [ATP]. 3. At each total [ATP], elevation of the free [Mg2+] from the normal resting level of 1 mM to 3 mM, caused a similar reduction in depolarization-induced Ca2+ release. This effect was not due to the concomitant decrease in free [ATP] and occurred independently of the reduction in Ca2+ release observed at very low total [ATP]. 4. These results show myoplasmic [ATP] modulates depolarization-induced Ca2+ release. Thus, if the total [ATP] near the Ca2+ release channels becomes locally depleted in severely fatigued muscle fibres, it may contribute, in combination with an accompanying increase in free [Mg2+], to the reported reduction in Ca2+ release.