FK506 binds to the members of the FK506-binding protein (FKBP) subfamily of immunophilins inhibiting their peptidyl-prolyl isomerase (PPI) activity, which has been shown to be required to modulate the permeability of endoplasmic reticulum-resident calcium channels, like IP3R and RYR (Adams et al., 2005; Kumar et al., 2005; MacMillan et al., 2008). Here we investigate the role of FKBPs during the activation of SOCE in human platelets. Human platelets were obtained from healthy volunteers according to the Declaration of Helsinki as previously described (Rosado and Sage, 2000). Incubation of fura 2-loaded platelets with FK506 for 5 min modified calcium release from intracellular pools and SOCE in human platelets. Low FK506 concentrations (up to 10 μM) increased SOCE activation induced either with thapsigargin (TG, 200 nM) or thrombin (Thr, 0.1 U/ml), meanwhile higher FK506 concentrations (50 μM) significantly reduced SOCE (P<0.05; Student,s t test). Platelets incubation with 2-APB (100 μM) for 30 min at 370C attenuated the increased calcium release evoked by FK506; although an alternative mechanism of calcium release sensible to FK506 in human platelets still remained active. Human platelets incubation for 30 min with the calcineurin (CNa) antagonist cypermethrin (100 nM; MacMillan et al., 2008) or for 5 min with cyclosporin A (50 μM; Adams et al., 2005) reduced TG-evoked SOCE, which indicates that CNa activity is required for the activation of SOCE. Furthermore, inhibition of FKBPs PPI activity by incubation for 30 min with rapamycin (500 nM), which does not affect CNa activity, also reduced significantly TG-induced SOCE. Co-immunoprecipitation experiments revealed that FK506 reduced Thr- and TG-induced IP3R II/TRPC1 coupling, which plays an important role in the maintenance of SOCE. The association between TRPC1 and FKBP12 was sensitive to FK506 and rapamycin. In contrast, the coupling of TRPC1 with CNa was affected by FK506 but insensitive to rapamycin. In summary, here we show for the first time that the activity of the immunophilin subfamily FKBP is important for the activation of SOCE by both CNa-dependent and -independent pathways in human platelets.