To examine whether the calpain-calpastatin system is activated during the calcium paradox in the isolated perfused pigeon heart, we separated the protease from its inhibitor calpastatin and studied its kinetic properties. The protease exhibits kinetic properties similar to those of mammalian m-calpains. Ca(2+) requirements for half and maximum activities are 220 microM and 2 mM, respectively. In the absence of Ca(2+) the protease is strongly activated by Mn(2+) or Sr(2+). In the presence of Ca(2+), Mn(2+) and Sr(2+) exhibit a synergistic effect; Mg(2+) and Ba(2+) have no effect, whereas Co(2+), Ni(2+) and Cd(2+) completely inhibit its activation. Furthermore, we measured the activity of calpain and calpastatin under either conditions inducing a calcium paradox, or protecting the heart against this phenomenon. Although the calpain/calpastatin ratio is lowered during Ca(2+) depletion, during Ca(2+) repletion it is markedly inverted. Calpain activation during reperfusion is inhibited by the presence of 200 microM Mn(2+) or Ba(2+), in the Ca(2+)-free medium. Gel filtration of calpastatin, isolated from either untreated hearts or during Ca(2+) depletion, produces two main peaks of ñ150 and 40 kDa of molecular mass, respectively, whereas calpastatin isolated during the 2(nd) min of reperfusion appears to be shifted to the 150 kDa form. All the above data suggest that this system may be involved in the induction of the calcium paradox in pigeon heart.