At the end of a long bout of activity, Ca²⁺ release from the sarcoplasmic reticulum (SR) decreases. Two mechanisms that may underlie the decline in SR Ca²⁺ release are 1. feedback of Ca²⁺ on the ryanodine receptor to decrease SR Ca²⁺ release or 2. precipitation of inorganic phosphate (Pi) and Ca²⁺ in the SR which reduces the free SR [Ca²⁺] available for release. Recently, N-benzyl-p-toluene sulphonamide (BTS) has been found to act as a potent and specific inhibitor of muscle myosin and also to have no effect on the Ca²⁺ transients associated with twitches (Cheung et al., 2002). In the presence of BTS, repeated electrical stimulation of muscle would be expected to result in decreased usage of ATP by the cross-bridges, reduced [Pi] and slower formation of Ca²⁺-Pi precipitates in the SR. This study was approved by the Stockholm North local ethical committee. Adult NMRI mice were killed by rapid neck disarticulation. Single intact fibres were dissected from the flexor digitorum brevis, a fast toe muscle. Fibres were stimulated to contract using 70 Hz, 350 ms tetani and fatigue was induced by applying stimuli at 2 s intervals until force was down to 40% of its starting value. Isometric tetanic force and [Ca²⁺]_i (monitored with the fluorescent Ca²⁺ indicator indo-1) were measured. All experiments were performed at room temperature (24 − 26 °C). After 10 min in 10 μM BTS, force was reduced by more than 90% from 393 ± 14 to 36 ± 14 kPa (mean ± S.E.M., n = 5). However, tetanic [Ca²⁺]_i was not significantly altered being 1.35 ± 0.27 μM and 1.31 ± 0.22 μM in the absence and presence of BTS respectively. The time course of the Ca²⁺ transient was not altered by BTS. The effects of BTS were essentially reversible with force recovering to about 90% of its starting value after 20 min washout. These results emphasize that BTS blocks myosin and has no effect on SR Ca²⁺ release. Fibres were fatigued either in the absence or presence of 10 μM BTS. Force was reduced to 40% after 61 ± 15 tetani (n = 8) and 166 ± 12 tetani (n = 9) in the absence and presence of BTS respectively. Tetanic [Ca²⁺]_i declined more rapidly in the absence of BTS than in its presence. These results suggest that in intact fibres, elevated [Ca²⁺]_i does not have adverse feedback effects on SR Ca²⁺ release. Instead the decline of tetanic [Ca²⁺]_i may be related to metabolic changes e.g. slower formation of Ca²⁺-Pi precipitates in the SR.