We investigate signal transduction pathways that mediate the actions of hormones, growth factors, and neurotransmitters in mammalian cells, with special emphasis on the role of phosphoinositide-derived messengers. Phosphoinositides constitute a small fraction of the cellular phospholipids but play critical roles in the regulation of many signaling protein complexes that assemble on the surface of cellular membranes and are intracellular lipid messengers controlling a variety of cellular functions. Phosphoinositides regulate protein kinases and GTP–binding proteins as well as membrane transporters, including ion channels, thereby controlling many cellular processes such as proliferation, apoptosis, metabolism, cell migration, and differentiation. We focus on the phosphatidylinositol 4 (PtdIns4)–kinases (PI4Ks), a family of enzymes that catalyze the first committed step in polyphosphoinositide synthesis. Current work aims to: (1) understand the function and regulation of several PI4Ks involved in the control of cellular signaling and trafficking pathways; (2) find specific inhibitors for the individual PI4Ks; (3) define the molecular basis of PtdIns4P–regulated pathways through identification of PtdIns4P–interacting molecules; (4) develop tools to analyze inositol lipid dynamics in live cells; and (5) determine the importance of the lipid-protein interactions in the activation of cellular responses by G protein–coupled receptors and receptor tyrosine kinases.