Two separate Ca2+ stores have been reported in human platelets: the dense tubular system (DTS) and lysosome-like acidic organelles. Recent work has reported that Ca2+ release from the DTS is mediated by the generation of inositol 1,4,5-trisphosphate, whereas Ca2+ efflux from the acidic stores is mostly linked to nicotinic acid adenine dinucleotide phosphate. Platelet agonists release Ca2+ selectively from one or both stores, which provides additional insight into the complexity of Ca2+ signaling and the cellular functions activated. Here, we review the role of multiple Ca2+ mobilizing messengers and Ca2+ stores in the activation of specific functions in platelets in response to different physiologic agonists.