The roles of lipids expand beyond the basic building blocks of biological membranes. In addition to forming complex and dynamic barriers, the thousands of different lipid species in the cell contribute to essentially all the processes of life. Specific lipids are increasingly identified in cellular processes, including signal transduction, membrane trafficking, metabolic control and protein regulation. Tight control of their synthesis and degradation is essential for homeostasis. Most of the lipid molecules in the cell originate from a small number of critical intermediates. Thus, regulating the synthesis of intermediates is essential for lipid homeostasis and optimal biological functions. Cytidine diphosphate diacylglycerol (CDP-DAG) is an intermediate which occupies a branch point in lipid metabolism. CDP-DAG is incorporated into different synthetic pathways to form distinct phospholipid end-products depending on its location of synthesis. Identification and characterization of CDP-DAG synthases which catalyze the synthesis of CDP-DAG has been hampered by difficulties extracting these membrane-bound enzymes for purification. Recent developments have clarified the cellular localization of the CDP-DAG synthases and identified a new unrelated CDP-DAG synthase enzyme. These findings have contributed to a deeper understanding of the extensive synthetic and signaling networks stemming from this key lipid intermediate.