1. This study examined whether reduced [ATP], raised [Mg2+] and the presence of the metabolites AMP and inosine monophosphate (IMP) affected depolarization-induced Ca2+ release from the sarcoplasmic reticulum (SR) in mechanically skinned skeletal muscle fibres of the rat. The amount of Ca2+ released was determined from the extent of SR Ca2+ depletion following a depolarization in the specified conditions with 2 mM free EGTA present to chelate released Ca2+. 2. In the presence of 8 mM total ATP and 1 mM free Mg2+, most of the SR Ca2+ could be released by a single (2-3 s) depolarization. Paired comparisons in the same fibres showed that raising the [Mg2+] from 1 to 3 mM reduced the total amount of Ca2+ released by a single depolarization by approximately 40 %. At 1 mM Mg2+, lowering the [ATP] to 0.5 mM did not cause a detectable change in the total amount of Ca2+ released, but when the release rate was reduced by the presence of 3 mM Mg2+, lowering the [ATP] to 0.5 mM resulted in a further ( approximately 20 %) reduction in the total amount of Ca2+ released. 3. At 1 mM Mg2+ and 0.5 mM ATP, neither the presence of 3 mM AMP alone nor 3 mM AMP plus 3 mM IMP caused a significant change in total Ca2+ release. Furthermore, at 1 mM Mg2+, the combined effect of lowering the [ATP] from 8 to 0.5 mM and simultaneously adding 3 mM AMP and 3 mM IMP did not significantly alter total Ca2+ release. However, when Ca2+ release was already reduced by the presence of 3 mM Mg2+ and 0.5 mM ATP (to approximately 50 %), addition of 3 mM AMP and 3 mM IMP significantly reduced the amount of Ca2+ released a further 2-fold. 4. These results show that depolarization-induced Ca2+ release in mammalian muscle fibres is modulated by the concentration of ATP and its metabolic products, as well as by the free [Mg2+]. Consequently, the (reversible) reduction in Ca2+ release occurring in a muscle fibre after prolonged exercise could result not only from raised [Mg2+] but also from a severe reduction in [ATP] locally near the Ca2+ release channels, with the accompanying build-up of AMP and IMP further exacerbating this effect.