The aim of this study was to examine whether prolonged low-frequency force depression (PLFFD) that occurs in situ is the result of decreased myofibrillar Ca(2+) sensitivity and/or reduced sarcoplasmic reticulum (SR) Ca(2+) release. Intact rat gastrocnemius muscles were electrically stimulated via the sciatic nerve until force was reduced to ~50% of the initial and dissected 30 min following the cessation of stimulation. Skinned fibre and whole muscle analyses were performed in the superficial region composed exclusively of type IIB fibres. Fatiguing stimulation significantly reduced the ratio of force at low frequency to that at high frequency to 65% in skinned fibres (1 vs. 50 Hz) and 73% in whole muscles (20 vs. 100 Hz). In order to evaluate changes in myofibrillar Ca(2+) sensitivity and ryanodine receptor caffeine sensitivity, skinned fibres were activated in Ca(2+)- and caffeine-containing solutions, respectively. Skinned fibres from fatigued muscles displayed decreased caffeine sensitivity together with increased myofibrillar Ca(2+) sensitivity. Treatment with 2,2'-dithiodipyridine and reduced glutathione induced a smaller increase in myofibrillar Ca(2+)sensitivity in fatigued than in rested fibres. In fatigued muscles, S-glutathionylation of troponin I was increased and submaximal SR Ca(2+) release, induced by 4-chloro-m-cresol, was decreased. These findings suggest that in the early stage of PLFFD that occurs in fast-twitch muscles of exercising animals and humans, S-glutathionylation of troponin I may attenuate PLFFD by increasing myofibrillar Ca(2+) sensitivity and that under such a circumstance, PLFFD may be ascribable to failure of SR Ca(2+) release.