Store-operated calcium entry (SOCE) is an important pathway for calcium influx in human platelets. Previous studies have demonstrated a role for extracellular signal-regulated kinase (ERK) in the activation of SOCE in these cells (Rosado & Sage, 2001). Components of the ERK cascade involved in SOCE are uncharacterised. One candidate is m-calpain which has been shown in other cell types to be activated, at least in part, by ERK phosphorylation (Glading et al., 2004). Calpain has a role in many aspects of platelet activation including secretion, spreading and aggregation (Croce et al., 1999). Here we have studied the effects of calpain inhibition on SOCE in human platelets. Fura-2-loaded platelets were preincubated at 37°C for 10 minutes with various concentrations of Calpain Inhibitor III (C3) and then stimulated using 200nM thapsigargin (TG) in the presence of 100μM EGTA for 3 minutes before addition of 1.6mM SrCl₂. Sr²⁺ is a poor substrate for the plasma membrane Ca²⁺-ATPase (PMCA) and so was used to estimate unidirectional divalent cation influx. C3 caused a dose-dependent inhibition of Sr²⁺ entry with 300μM C3 reducing entry to 16 ± 2 % of control (mean ± S.E.M., n=7; p<0.005). There was also an apparent potentiation of TG-evoked Ca²⁺ release, probably attributable to an inhibition of the PMCA. Similar results were obtained with the mechanistically different calpain inhibitor, PD150606. Neither inhibitor acted as a store-operated channel blocker. Thrombin-evoked Ca²⁺ signalling was also significantly inhibited by C3 preincubation. Platelet calpain activity was monitored by loading washed cells with the cell permeable, fluorogenic calpain substrate t-BOC-Leu-Met CMAC (100μM for 40 minutes at 37°C). Cells were then preincubated with 300μM C3 or the vehicle and the rate of calpain activity was measured for 20 seconds after stimulation with 200nM TG or vehicle in the presence of 200μM EGTA in a stopped-flow fluorimeter. TG increased calpain activity to 143 ± 13 % of basal (mean ± S.E.M., n=7; p<0.01). The TG-evoked increase was reduced to 124 ± 10 % of basal after preincubation with C3 and was closely temporally matched to the onset of TG-evoked Ca²⁺ release. We have demonstrated that TG-evoked Ca²⁺ store depletion rapidly induces an increase in calpain activity that plays a role in the activation of SOCE in human platelets. This is the first report of the intracellular measurement of calpain activity in human platelets and is also the first demonstration of a role of a protease in SOCE.