The zeta-form 14-3-3 protein (14-3-3zeta) regulates protein kinases and interacts with several signaling molecules. We reported previously that a platelet adhesion receptor, glycoprotein (GP) Ib-IX, was associated with a 29-kDa protein with partial sequences identical to 14-3-3zeta. In this study, the interaction between GPIb-IX and recombinant 14-3-3zeta is reconstituted. Further, we show that the 14-3-3zeta binding site in GPIb is within a 15 residue sequence at the C terminus of GPIb-alpha, as indicated by antibody inhibition and direct binding of 14-3-3zeta to synthetic GPIb-alpha cytoplasmic domain peptides. The 14-3-3zeta binds to recombinant wild type GPIb-IX but not to the GPIb-alpha mutants lacking C-terminal 5 or more residues, suggesting that the C-terminal 5 residues of GPIb-alpha are critical. Similarity between the GPIb-alpha C-terminal sequence and the serine-rich regions of Raf and Bcr kinases suggests a possible serine-rich recognition motif for the 14-3-3 protein.