Summary. Background: Platelets have three major types of secretory organelles: lysosomes, dense granules, and α-granules. α-Granules contain several adhesive proteins involved in hemostasis, as well as glycoproteins involved in inflammation, wound healing, and cell–matrix interactions. This article represents the first effort to define the platelet α-granule proteome using mass spectrometry (MS). Methods: We prepared a subcellular fraction enriched in intact α-granules from human platelets using sucrose gradient ultracentrifugation. α-Granule proteins were separated and identified using sodium dodecylsulfate polyacrylamide gel electrophoresis and liquid chromatography–tandem MS. Results: In the sucrose fraction enriched in α-granules, we identified 284 non-redundant proteins, 44 of which appear to be new α-granule proteins, on the basis of a literature review. Immunoelectron microscopy confirmed the presence of Scamp2, APLP2, ESAM and LAMA5 in platelet α-granules for the first time. We identified 65% of the same proteins that were detected in the platelet releasate (J. A. Coppinger et al. [Blood 2004;103: 2096–104]) as well as additional soluble and membrane proteins. Our method provides a suitable tool for analyzing the granule proteome of patients with storage pool deficiencies.