Skeletal muscle fibres contain ubiquitous and muscle-specific calcium-dependent proteases known as calpains. During normal activity, intracellular [Ca(2+)] in muscle fibres increases to high levels ( approximately 2-20 microm), and it is not apparent how this can be reconciled with the activation properties of the calpains. Calpains evidently do not cause widespread proteolytic damage within muscle fibres under normal circumstances, but do have a role in necrosis in dystrophic muscle fibres. In this study, we examined the in situ localization and regulation of calpains in muscle fibres in order to identify how they are attuned to normal function. The sarcolemma of individual muscle fibres of the rat was removed by microdissection (fibre 'skinning') in order to determine the compartmentalization and diffusibility of the two most Ca(2+)-sensitive calpains, mu-calpain and calpain-3, and to permit precise manipulation of cytoplasmic [Ca(2+)] under physiological in situ conditions. Passive force production in stretched fibres, which indicates the patency of the important elastic structural protein titin, was used as a sensitive assay of the amount of diffusible proteolytic activity in individual fibre segments and in muscle homogenates at set [Ca(2+)]. All calpain-3 is bound tightly within a fibre, whereas most mu-calpain ( approximately 0.2 microm) is initially freely diffusible in the cytoplasm at resting [Ca(2+)] but binds within seconds at high [Ca(2+)]. [Ca(2+)] has to be raised to >/= 2 microm for >/= 1 min to initiate detectable autolysis of mu-calpain and to activate appreciable proteolytic activity. If the [Ca(2+)] is raised sufficiently for long enough to initiate substantial autolysis of mu-calpain, the Ca(2+) sensitivity of the proteolytic activity is greatly increased, and it remains active even at 300 nm Ca(2+), with activity only ceasing if the [Ca(2+)] is decreased to approximately 50 nm Ca(2+), close to the normal resting [Ca(2+)]. These findings on the Ca(2+)- and time-dependent binding, autolytic and proteolytic properties of mu-calpain under physiological conditions demonstrate how it is precisely attuned to avoid uncontrolled proteolytic activity under normal circumstances, and indicate why it could lead to substantial proteolytic damage if resting or localized [Ca(2+)] is elevated, as is likely to occur after eccentric contraction and in dystrophic muscle.