Significant roles for phosphatidylinositols (PtdIns) in membrane traffic have emerged in addition to their originally described function as intracellular second messengers. Distinct PtdIns species have been implicated in regulating interactions between the lipid bilayer and protein machinery involved in vesicle budding and traffic as well as in modulating cytoskeletal dynamics that affect transport kinetics. Efficient regulation of PtdIns production requires spatial and temporal control of PtdIns metabolism, and this is mediated in part by the subcellular distribution of phosphatidylinositol kinases (PIKs) and phosphatases. We are interested in the role of PtdIns metabolism in polarized biosynthetic delivery, and to this end, we have examined the effect of expressing individual PIKs and PtdIns phosphatases on lipid modulation and membrane traffic in a polarized renal epithelial cell system. Our studies have focused primarily on phosphatidylinositol 4- and 5-kinases, which sequentially metabolize PtdIns to phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Whereas the effects of PI4Ks on membrane traffic are most consistent with effects on the recruitment of trafficking machinery, overexpression of PI5K selectively stimulated surface delivery of an apical marker via a mechanism consistent with the involvement of actin comets. Our results suggest multiple mechanisms by which PtdIns synthesis and conversion differentially affect the surface delivery of apically- and basolaterally-destined proteins in polarized cells.