The current status of the biochemistry of phospholipid biosynthesis is presented. The review focuses on the identification and characterization of molecular tools such as purified enzymes and cloned genes and cDNAs for those enzymes. The enzymes discussed are those involved in the biosynthesis of the major phospholipid classes, namely, phosphatidate, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, phosphatidylinositol and its phosphorylated derivatives, and cardiolipin. The review centers on the pathways in mammals and yeast. Novel genetic approaches used to delineate pathways and clone cDNAs are discussed. The regulatory roles played by some of the enzymes involved in controlling the biosynthetic pathways are presented.