Like any other eukaryotic cells, the membrane of Toxoplasma gondii contains different phospholipids. Few phospholipids are generic such as phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), phosphatidylinositol (PtdIns) and sphingomyelin, whereas others are rather exclusive to certain parasitic protists, e.g., phosphatidylthreonine (PtdThr). PtdThr is a natural structural analog of otherwise-universal PtdSer. T. gondii harbors de novo pathways to synthesize several phospholipids but at the same time, it can also salvage selected lipids from its host cell. Our previous works have revealed the important role of PtdThr in parasite motility, and locomotion-dependent invasion and egress events, indicating a connection between PtdThr and calcium signaling. Similarly, we found a number of P4-ATPAse proteins potentially involved in flipping of PtdSer and PtdThr. Phospholipid biosynthesis takes place at different subcellular locations in the parasite. PtdThr, PtdCho, and PtdSer are produced in the endoplasmic reticulum (ER), whereas PtdEtn is made at many places including the ER, mitochondrion and parasitophorous vacuole (PV). In the current project, we have investigated the role of PtdThr and PtdIns in parasite growth and virulence. At the same time, the grant has helped us understand the role of flippase in PtdSer flipping and calcium homeostasis by SERCA. We have identified five P4-ATPases (P4-ATPase1-5) in Toxoplasma and characterized them. Most of the work is published and a manuscript is under submission.