The intracellular parasite Toxoplasma gondii develops inside a parasitophorous vacuole (PV) that derives from the host cell plasma membrane during invasion. Previous electron micrograph images have shown that the membrane of this vacuole undergoes an extraordinary remodeling with an extensive network of thin tubules and vesicles, the intravacuolar network (IVN), which fills the lumen of the PV. While dense granule proteins, secreted during and after invasion, are the main factors for the organization and tubulation of the network, little is known about the source of lipids used for this remodeling. By selectively labeling host cell or parasite membranes, we uncovered evidence that strongly supports the host cell as the primary, if not exclusive, source of lipids for parasite IVN remodeling. Fluorescence recovery after photobleaching (FRAP) microscopy experiments revealed that lipids are surprisingly dynamic within the parasitophorous vacuole and are continuously exchanged or replenished by the host cell. The results presented here suggest a new model for development of the parasitophorous vacuole whereby the host provides a continuous stream of lipids to support the growth and maturation of the PVM and IVN.Toxoplasma gondii is an apicomplexan parasite that infects almost any warm-blooded animal. This ubiquitous parasite is able to proliferate inside the host cell within a unique and dynamic parasitophorous vacuole (PV). This vacuole is established during invasion initially as an invagination of the host cell plasma membrane that surrounds the invading parasite and then pinches off to create a PV (18). During this process, parasite secretory organelles, called dense granules, discharge specific protein factors (GRAs) that remodel the vacuole into a sheltered niche for the parasite.Toxoplasma's ability to acquire essential nutrients from its environment is fundamental to its intracellular lifestyle. It has been postulated that Toxoplasma gains access to various host metabolites (Ͻ1,300 Da) via diffusion through small pores in the PV membrane (PVM) (14). While no direct connection between the PV and the host vesicular transport system has been observed (13), Toxoplasma incorporates low-density lipoprotein (LDL)-derived cholesterol from host endolysosomes (4). Host mitochondria are also recruited efficiently to the cytosolic face of the PV soon after invasion, allowing Toxoplasma to scavenge host lipoic acid (5, 16).In addition to nutrient acquisition from the host cytosol, Toxoplasma facilitates its intracellular lifestyle by actively remodeling the PV. The purpose of this remodeling and the origin of the lipid constituents it uses have not been established. Electron micrographs of the Toxoplasma PV show membrane-like material within the PV and close to the posterior end of the parasites at very early times during invasion (15). A complex of secreted GRA2/GRA4/GRA6 proteins interacts with this material and seems to be required for its reorganization and tubulation (9, 12). Later during infection, similar thin tubul...