The glycoprotein (GP) Ib-IX-V complex constitutively expressed on the platelet plasma membrane mediates initial adhesion of circulating platelets to vessel wall matrix at high shear, and shear-induced platelet aggregation. In both cases, this involves binding of GP Ib-IX-V to the adhesive glycoprotein, von Willebrand Factor (vWF). vWF binding to GP Ib-IX-V rapidly induces platelet activation, leading to cytoskeletal rearrangement, shape change, and secretion that enables αIIbβ3 integrin (GP Ilb-IIIa)-dependent platelet aggregation. All these events are critical in (patho)physiological thrombus formation. The recent discovery that the signaling protein, 14-3-3 ζ, copurifies with the GP Ib-IX complex (minus GP V) [Du, X., Harris, S. J., Tetaz, T. J., Ginsberg, M. H., and Berndt, M. C. (1994) J. Biol. Chem. 269, 18287-18290] indicated a potential mechanism for vWF-dependent signaling. The aim of the present study was to identify discrete amino acid sequences that bind 14-3-3 ζ within the cytoplasmic domain of the receptor. As an initial screening assay, overlapping synthetic peptides based on the cytoplasmic domains of GP Ibα (100 residues), GP Ibβ (34 residues), GP IX (5 residues), and GP V (16 residues) were immobilized and assessed for the ability to bind purified 14-3-3 ζ. The C-terminal sequence GHSL of GP Ibα was identified as one 14-3-3 ζ interactive sequence, consistent with previous results [Du, X., Fox, J. E., and Pei, S. (1996) J. Biol. Chem. 271, 7362-7367]. Binding of 125I-labeled 14-3-3 ζ to GHSL-containing peptides was inhibitable by unlabeled 14-3-3 ζ and by anti-14-3-3 ζ IgG. Ala-walking through the GHSL sequence suggested all residues were necessary for optimal binding. In addition, 14-3-3 bound with lower affinity to a peptide based on the central region of the GP Ibα cytoplasmic domain (Arg-557-Gly-575), whereas peptide sequences within the cytoplasmic domains of GP Ibβ (Arg-160-Arg-175) and GP V (Lys-529-Gly-544) bound 14-3-3 ζ with comparable affinity to the GHSL- containing peptide. Soluble GHSL-containing peptides, GP Ibβ- and GP V- based peptides semidissociated 14-3-3 from GP Ib-IX-V or GP Ib-IX in platelet extracts as analyzed by immunoprecipitation, suggesting these sequences, at least partially, mediate the GP lb-IX-V- 14-3-3 ζ interaction in cells. Further, phosphorylation of the GP lbβ peptide at a site corresponding to a protein kinase A phosphorylation site (Ser-166) enhanced the affinity of 14- 3-3 ζ binding by approximately 8-fold, suggesting phosphorylation as a potential mechanism for regulating 14-3-3 ζ association with the GP Ib-IX-V complex.