Aim The study was undertaken to explore whether shortening of striated muscle during activity is associated with release of bound Ca2+ into the myofibrillar space as has previously been proposed in order to explain the depressant effect of active shortening. Methods The experiments were carried out on single muscle fibres isolated from the anterior tibialis muscle of Rana temporaria. The fibres were loaded with the calcium sensitive indicator Fluo‐3. The fibres, stimulated to produce a partially fused isometric tetanus, were subjected to a shortening ramp or, alternatively, to a stretch ramp during activity while force, fibre length, sarcomere length and the Fluo‐3 signal were recorded. Results A shortening ramp performed during a partially fused tetanus caused an increase in the myofibrillar free calcium concentration and produced, simultaneously, a decrease in active force. The isometric force recovered gradually after the shortening ramp, while the intracellular Ca2+ concentration stayed above the control level during the remainder of the stimulation period. A stretch ramp applied during a partially fused tetanus caused a considerably smaller change in the myofibrillar Ca2+ concentration. Conclusion The results provide evidence that the myosin cross‐bridges interact with the calcium binding sites on the thin filaments during active shortening, causing sustained release of calcium and reduced contractile strength.