The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyzes and thereby inactivates the second messenger molecules inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate in a signal terminating reaction. Recent studies have shown that the platelet protein pleckstrin forms a complex with the 43 kDa 5-phosphatase and activates Ins(1,4,5)P3 hydrolysis 2-fold [Auethavekiat, V., Abrams, C. S., and Majerus, P. W. (1997) J. Biol. Chem. 272, 1786-1790]. We now show that another platelet protein, 14-3-3ζ, forms a complex with the 43 kDa 5- phosphatase and thereby activates the hydrolysis of Ins(1,4,5)P3. Both pleckstrin and 14-3-3ζ contain one or more pleckstrin-homology domains, both are present in platelet cytosol, and both dimerize and form complexes with other signalling proteins. Purified platelet pleckstrin and 14-3-3ζ enhanced the rate of the hydrolysis of Ins(1,4,5)P3 by the 43 kDa 5-phosphatase 1.9- and 3.8-fold, respectively, but did not activate the 75 kDa 5-phosphatase. We have demonstrated that the mechanism of 5-phosphatase activation by 14-3- 3ζ results from specific complex formation between the 43 kDa 5-phosphatase and 14-3-3ζ. Recombinant 43 kDa 5-phosphatase bound to recombinant glutathione S-transferase (GST)/14-3-3ζ fusion protein, but not GST alone, immobilized on glutathione-Sepharose. A potential 14-3-3 binding motif was located in the 43 kDa, but not the 75 kDa, 5-phosphatase. The motif '363RSESEE' is present in close proximity to the proposed catalytic domain of the 43 kDa 5-phosphatase. A synthetic peptide corresponding to the putative 14-3-3 binding motif demonstrated specific, saturable binding to purified 125I-14-3-3, with a K(d) of 92 nM. In addition, platelet cytosolic 5-phosphatase bound to recombinant 14-3-3ζ immobilized on glutathione-Sepharose. Thus, 14-3-3ζ serves in human platelets to activate the 43 kDa 5-phosphatase and may thereby function to prevent generation of Ins(1,4,5)P3 -mediated calcium release in unstimulated platelets.