This chapter discusses the metabolism of ether-linked diglycerides in brain and myocardium. The fundamental differences between ether-linked and diacyl phospholipids are also described. The chapter also discuses methods for quantitating ether-linked diglycerides in mammalian cells and to assess the biochemical pathways responsible for ether-linked diglyceride metabolism utilizing synthetic ether-linked diglycerides purified by high-performance liquid chromatography (HPLC). The synthesis of specific molecular species of ether-linked diglycerides is accomplished through utilization of appropriate subclasses or individual molecular species of lysophospholipid or monoradylglycerol precursors. These precursors can be specifically acylated with the desired fatty acid at the sn-2 position (for lysophospholipids) or sequentially acylated at the sn-3 and sn-2 positions (for monoradylglycerols) prior to their enzymatic conversion to ether-linked diglycerides. Ether-linked diglycerides can subsequently be purified by thin-layer chromatography (TLC), straight-phase HPLC, or both, to yield substantial amounts (10–100 mg) of homogeneous ether-linked diglycerides.