We developed a microsome-based, cell-free system that assembles newly formed triglyceride (TG) into spherical lipid droplets. These droplets were recovered in the d < 1.055 g/ml fraction by gradient ultracentrifugation and were similar in size and appearance to those isolated from rat adipocytes and 3T3-L1 cells. Caveolin 1 and 2, vimentin, adipocyte differentiation-related protein, and the 78-kDa glucose regulatory protein were identified on the droplets from the cell-free system. The caveolin was soluble in 1% Triton X-100, as was the caveolin on lipid droplets from 3T3-L1 cells. The lipid droplets from the cell-free system, like those from 3T3-L1 cells, contained TG, diacylglycerol, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. The assembly of these TGcontaining structures was dependent on the rate of TG biosynthesis and required an activator present in the 160,000 ؋ g supernatant from homogenized rat adipocytes. The activator induced phospholipase D (PLD) activity, and its effect on the release of the TG-containing structures from the microsomes was inhibited by 1-butanol (but not 2-butanol) or 2,3-diphosphoglycerate. The activator could be replaced by a constitutively active PLD or phosphatidic acid. These results indicate that PLD and the formation of phosphatidic acid are important in the assembly of the TG-containing structures.Within the cell, triglycerides (TG) 1 are stored in cytosolic lipid droplets. Little is known about how these structures are assembled. Several proteins have been identified on their surface, including adipocyte differentiation-related protein (ADRP or adipophilin), perilipins (reviewed in Refs. 1 and 2), and caveolin (see Refs. 3-5 and reviewed in Refs. 2 and 6), but their roles in lipid droplet assembly are unknown.ADRP and perilipins are abundant on lipid droplets. ADRP is found mostly on smaller droplets; when overexpressed, it stimulates lipid droplet formation (7), suggesting a role in the assembly process. Perilipins are present on large lipid droplets (7) and have a central role in the turnover of TG within these structures (1, 8, 10), perhaps affording protection against hormone-sensitive lipase (11). Perilipin-null mice have smaller lipid droplets and a higher rate of basal lipolysis than wild-type mice and are resistant to diet-induced obesity (12).Caveolin is a 21-kDa membrane protein with a hairpin structure whose N and C termini face the cytosol (13, 14). In mammals, there are three caveolin genes. Caveolin 1 and 2 are expressed in adipocytes, whereas caveolin 3 is present in muscle. Caveolin plays a key role in intracellular lipid transport (15, 16), binding fatty acid (17), and transporting cholesterol (16) in a manner reminiscent of the way plasma apolipoproteins transport lipids in the blood (16,18).Phospholipase D (PLD) catalyzes the conversion of phosphatidylcholine to phosphatidic acid (PA) and appears to be important in intracellular transport and sorting processes (19 -22), either as an intracellular messenger or as a cone-shaped l...