Human platelets were disrupted using nitrogen cavitation and fractionated on sucrose density gradients to permit isolation of α-granules, the major secretory granule of platelets. Membrane proteins prepared from intact α-granules by alkali extraction were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose and the blot probed for the presence of GTP-binding proteins using [α- 32P]GTP. Two low molecular mass GTP-binding proteins with molecular mass of 27 and 24 kDa, respectively, were identified on the α-granule membrane. In contrast to the 27-kDa protein which was present in significant amounts in the plasma membrane-enriched fraction, the 24-kDa protein showed a preferential association with the α-granule membrane. On immunoblotting with specific antiserum, the 24-kDa GTP-binding protein was found to be distinct from rab3A. To the best of our knowledge, the present report represents the first identification of low molecular mass GTP-binding proteins associated with a platelet secretory granule.