To clarify the physiological role of calcium-activated neutral protease (CANP) in human platelets, we loaded the platelets with a Ca2+-sensitive fluorescent dye, fura-2, and measured the degree of aggregation, cytosolic calcium ion concentration ([Ca2+]1), and proteolysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). At physiological concentration of Ca2+(1 mM) in the incubation medium, [Ca2+], was below 0.5 μM and platelet aggregation was not shown. Ionomycin (0.15 μM) or collagen (50 μg/ml), but not ADP (10 fiM), sharply enhanced the [Ca2+]1to near 1 μM and caused the aggregation. A calcium entry blocker, verapamil, completely abolished both the [Ca2+]1rise and the aggregation. NCO-700, a membrane permeable inhibitor against cysteine proteases (including CANP), dose-dependently blocked the aggregation but did not change the [Ca2+]1transient. SDS-PAGE revealed that filamin, talin, and 70 kDa protein were specifically degraded when platelets were aggregated by ionomycin or collagen and that the proteolysis was not observed when the aggregation was blocked by verapamil or NCO-700. These data provided evidence that Ca2+entry exceeding 0.5 μM is essential, but not sufficient per se, and that activation of cysteine protease, most likely CANP, is involved in the platelet aggregation by collagen or calcium ionophore.