Mechanically skinned muscle fibres from the extensor digitorum longus (EDL) muscle of the rat were electrically stimulated in solutions mimicking the myoplasmic environment in the resting muscle fibre but containing different [ADP] of < 0.1 microm, 40 microm and 1.0 mm, to investigate the effects of myoplasmic ADP on the twitch response. The amplitude of the twitch response markedly and gradually decreased by 47 +/- 6% (n=9) as [ADP] was increased from < 0.1 microm to 40 microm without changing [Ca2+] in the myoplamsic solution (50 nm). The times for the twitch to rise from 10 to 90% (Trise,10-90) and to decrease from 90 to 10% (Tfall,90-10) initially increased by 8 and 21% and then decreased by 16 and 30% (compared to controls), respectively, at steady state. When [ADP] was raised from < 0.1 microm to 1.0 mm and fibres were electrically stimulated, the first response was biphasic and very prolonged (by at least a factor of 10) but of an amplitude similar to that in the control solution. The following twitch response and the steady state twitch responses were much reduced in size by about a factor of 6 and more prolonged by about 40% compared to control responses. All these ADP effects were fully reversible and appear to be predominantly due to several ADP-dependent alterations in SR Ca2+ handling properties (ADP-dependent decrease in SR Ca2+ capacity together with an increase in Ca2+ binding to the SR pump sites facing the myoplasm). The ADP-dependent effects on the contractile apparatus and Ca2+ regulatory system were relatively minor. Taken together, the results demonstrate that ADP accumulation is likely to play a crucial role in metabolic fatigue of skeletal muscle and can explain the marked reduction in the amplitude and the slower time course of the twitch response during fatigue as well as the elevation of myoplasmic [Ca2+] in fatigued fibres at rest.