Cells accumulate Ca2+ into agonist-sensitive acidic organelles, such as secretory granules and lysosome-related organelles, which express a vacuolar proton-ATPase (V-ATPase) responsible for the maintenance of a proton gradient across their membranes. Current evidence suggests that, in human platelets, acidic Ca2+ stores are involved in the activation of store-operated Ca2+ entry (SOCE) (Rosado et al., 2004), a mechanism for Ca2+ influx controlled by the filling state of the Ca2+ stores. Although STIM1 is considered the endoplasmic reticulum Ca2+ sensor, its presence and sensing role in the acidic stores have not been studied yet. Here we have investigated the expression of STIM1 in acidic Ca2+ stores and its association with plasma membrane Ca2+ channels upon acidic store depletion. Blood was drawn from volunteers with local ethical committee approval. Cytosolic Ca2+ concentration ([Ca2+]i) measurement, immunoprecipitation and Western blotting were performed as previously described (Redondo et al., 2006). Human platelets express STIM1 in lysosomes and lysosomal-related organelles, such as dense granules, isolated by immunomagnetic sorting. In dimethyl BAPTA-loaded cells, depletion of the acidic Ca2+ stores using the specific V-ATPase inhibitor, bafilomycin A1, or 2,5-di-(t-butyl)-1,4-hydroquinone (TBHQ), a SERCA3 inhibitor resulted in increased association between STIM1 and the capacitative Ca2+-selective channel Orai1. Furthermore, treatment with TBHQ produced time-dependent co-immmunoprecipitation of STIM1 or Orai1 with the TRPC proteins hTRPC1 and hTRPC6, which was found to occur independently of changes in [Ca2+]i. These results support the involvement of STIM1 in the activation of SOCE regulated by the acidic Ca2+ stores, and also the role of the channel proteins Orai1, hTRPC1 and hTRPC6 in Ca2+ entry after depletion of the acidic stores.