In the preceding paper, we reported that flexor digitorum brevis (FDB) muscle fibres from S100A1 knock‐out (KO) mice exhibit a selective suppression of the delayed, steeply voltage‐dependent component of intra‐membrane charge movement current termed Q_γ. Here, we use 50 μm of the Ca²⁺ indicator fluo‐4 in the whole cell patch clamp pipette, in addition to 20 mm EGTA and other constituents included for the charge movement studies, and calculate the SR Ca²⁺ release flux from the fluo‐4 signals during voltage clamp depolarizations. Ca²⁺ release flux is decreased in amplitude by the same fraction at all voltages in fibres from S100A1 KO mice compared to fibres from wild‐type (WT) littermates, but unchanged in time course at each pulse membrane potential. There is a strong correlation between the time course and magnitude of release flux and the development of Q_γ. The decreased Ca²⁺ release in KO fibres is likely to account for the suppression of Q_γ in these fibres. Consistent with this interpretation, 4‐chloro‐m‐cresol (4–CMC; 100 μm) increases the rate of Ca²⁺ release and restores Q_γ at intermediate depolarizations in fibres from KO mice, but does not increase Ca²⁺ release or restore Q_γ at large depolarizations. Our findings are consistent with similar activation kinetics for SR Ca²⁺ channels in both WT and KO fibres, but decreased Ca²⁺ release in the KO fibres possibly due to shorter SR channel open times. The decreased Ca²⁺ release at each voltage is insufficient to activate Q_γ in fibres lacking S100A1.