1. The sarcoplasmic reticulum (SR) Ca(2+) content (expressed in terms of endogenous SR Ca(2+) content under physiologically resting conditions and measured from caffeine-induced force responses) and the effective rates of the SR Ca(2+) pump and SR Ca(2+) leak (measured from the temporal changes in SR Ca(2+) content) were determined in mechanically skinned skeletal muscle fibres of the rat at different [ADP] (< 0.10 microM to 1.04 mM). 2. The estimated SR Ca(2+) pump rate at 200 nM Ca(2+) did not change when [ADP] increased from below 0.10 microM to 10 microM but decreased by about 30 % when [ADP] increased from 10 microM to 1.04 mM. 3. The rate constant of SR Ca(2+) leak increased markedly with rising [ADP] when [Ca(2+)] in solution was 200 nM (apparent dissociation constant Kd(ADP) = 64 +/- 27 microM). Decreasing the [Ca(2+)] in solution from 200 nM to < 10 nM significantly increased the leak rate constant at all [ADP]. The SR Ca(2+) leak rate constant could be significantly reduced by blocking the SR Ca(2+) pump with 2,5-di(tert-butyl)-1,4-hydroquinone (TBQ). 4. The decrease in the SR Ca(2+) pump rate and the increase in the rate constant of SR Ca(2+) leak when the [ADP] increased from < 0.10 microM to 1.04 mM caused a 4.4-fold decrease in SR Ca(2+) loading ability at 200 nM Ca(2+). 5. The results can be fully explained by a mechanism whereby the presence of ADP causes a marked increase in the ADP-sensitive fraction of the phosphorylated pump protein, which can act as a Ca(2+)-Ca(2+) exchanger and demonstrates that ADP is an important modulator of SR function in skeletal muscle.