The present report describes the radioactive labeling of polar lipids in in vitro cultures of Plasmodium falciparum as well as their extraction with organic solvents and their partial characterization by chemical and enzymatic methods. All substances detected could be cleaved by alkali, suggesting that they were esters rather than sphingolipids or compounds containing alkyl groups. Dolichol-cycle intermediates were not detected. Phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine were labeled by fatty acids and inositol or ethanolamine, respectively, confirming their de novo synthesis by the parasite. Metabolic labeling with glucosamine and cleavage by phosphatidylinositol-specific phospholipase C provided evidence of the formation of N-acetyl-glucosaminyl-phosphatidylinositol, an obligate precursor in the biosynthesis of glycosylphosphatidylinositol membrane anchors of proteins.