Current evidence suggests that phosphatidylinositol (PI) kinases and phosphatidylinositol transfer protein (PITP) are involved in driving vesicular traffic from yeast and mammalian trans-Golgi network (TGN). We have tested the interaction between these cytosolic proteins in an assay that measures the formation of constitutive transport vesicles from the TGN in a hepatocyte cell-free system. This reaction is dependent on a novel PI 3- kinase, and we now report that, under conditions of limiting cytosol, purified PI 3-kinase and PITP functionally cooperate to drive exocytic vesicle formation. This synergy was observed with both yeast and mammalian PITPs, and it also extended to the formation of PI 3-phosphate. These collective findings indicate that the PI 3-kinase and PITP synergize to form a pool of PI 3-phosphate that is essential for formation of exocytic vesicles from the hepatocyte TGN.