Calpains are Ca2+-activated proteases that are thought to be involved in muscle degenerative diseases such as Duchenne muscular dystrophy. Status and activity of calpains in adult muscle fibres are poorly documented. We report here in situ measurements of calpain activity in collagenase-isolated fibres from C57 mice and form two models of dystrophy: dystrophin-deficient mdx and calpain-3 knocked-out mice. Calpain activity was measured using a permeant, fluorogenic substrate and its Ca2+ dependence was studied. A 30-fold change of activity was observed between the lowest and the highest steady-state Ca2+ availability. Fast transient changes of [Ca2+]i induced by electrical stimulation or KCl-dependent depolarization were ineffective in activating calpain. Slow [Ca2+] transients, as elicited during depletion of Ca2+ stores, Ca2+ store repletion and hypo-osmotic swelling were able to activate calpain. On return to resting conditions, calpain activity recovered its basal rate within 10 min. In resting intact muscle, μ-calpain was predominantly in the 80 kDa native form, with a small fraction in the 78 kDa autolysed form. The latter is thought to be responsible for the activity measured in our conditions. Calpain activity in mdx fibres showed an average 1.5-fold increase compared to activity in C57 fibres. This activity was reduced by a 10-fold lowering of [Ca2+]o. Calpain-3-deficient fibres showed about the same increase, thus calpain-3 did not contribute to the activity measured here and calpain activation is not specific to dystrophin deficiency. In fibres from transgenic mice over-expressing calpastatin, a 40–50% reduction of calpain activity was observed, as with synthetic drugs (Z-Leu-Leu-CHO and SNT198438). We provide novel information on the physiological factors that control calpain activity in situ, particularly the effect of intracellular Ca2+ transients that occur in excitation–contraction coupling, Ca2+ store depletion and refilling, and activation of mechanosensitive Ca2+ channels.