GTP-binding proteins of molecular mass of 24-27 kDa were detected in the dense granule fraction of human platelets when nitrocellulose blots containing proteins separated by SDS-polyacrylamide gel electrophoresis were incubated with [alpha-32P]GTP. Further analysis, using isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis, resolved the dense granule 27 kDa and 24 kDa GTP-binding proteins into four distinct forms each. GTP-binding proteins in the total particulate fraction were resolved into seven 27 kDa and four 24 kDa forms. Immunoblotting with antiserum against known platelet low molecular mass GTP-binding proteins demonstrated that rap2 and G25K/CDC42Hs proteins, although present in platelets, were not detected in the dense granule fraction. However, ral was one of the proteins associated with dense granules. Association of specific low molecular mass GTP-binding proteins with dense granules suggests a potential role for these proteins in regulating the release of storage contents from this granule.