Phosphatidylinositol transfer protein a (PITPa) participates in the supply of phosphatidylinositol (PI) required for many cellular events including phospholipase C (PLC) ß and ? signaling by G-protein-coupled receptors and receptor-tyrosine kinases, respectively. Protein kinase C has been known to modulate PLC signaling by G-protein-coupled receptors and receptor-tyrosine kinases, although the molecular target has not been identified in most instances. In each case phorbol myristate acetate pretreatment of HL60, HeLa, and COS-7 cells abrogated PLC stimulation by the agonists formyl-Met-Leu-Phe, ATP, and epidermal growth factor, respectively. Here we show that phosphorylation of PITPa at Ser166 resulted in inhibition of receptor-stimulated PLC activity. Ser166 is localized in a small pocket between the 165-172 loop and the rest of the protein and was not solvent-accessible in either the PI- or phosphatidylcholine-loaded structures of PITPa. To allow phosphorylation at Ser166, a distinct structural form is postulated, and mutation of Thr59 to alanine shifted the equilibrium to this form, which could be resolved on native PAGE. The elution profile observed by size exclusion chromatography of phosphorylated PITPa from rat brain or in vitro phosphorylated PITPa demonstrated that phosphorylated PITPa is structurally distinct from the non-phosphorylated form. Phosphorylated PITPa was unable to deliver its PI cargo, although it could deliver phosphatidylcholine. We conclude that the PITPa structure has to relax to allow access to the Ser166 site, and this may occur at the membrane surface where PI delivery is required for receptor-mediated PLC signaling.