The addition of inositol to actively growing yeast cultures causes a rapid increase in the rate of synthesis of phosphatidylinositol and, simultaneously, triggers changes in the expression of hundreds of genes. We now demonstrate that the addition of inositol to yeast cells growing in the presence of choline leads to a dramatic reprogramming of cellular lipid synthesis and turnover. The response to inositol includes a 5-6-fold increase in cellular phosphatidylinositol content within a period of 30 min. The increase in phosphatidylinositol content appears to be dependent upon fatty acid synthesis. Phosphatidylcholine turnover increased rapidly following inositol addition, a response that requires the participation of Nte1p, an endoplasmic reticulum-localized phospholipase B. Mass spectrometry revealed that the acyl species composition of phosphatidylinositol is relatively constant regardless of supplementation with inositol or choline, whereas phosphatidylcholine acyl species composition is influenced by both inositol and choline. In medium containing inositol, but lacking choline, high levels of dimyristoylphosphatidylcholine were detected. Within 60 min following the addition of inositol, dimyristoylphosphatidylcholine levels had decreased from approximately 40% of total phosphatidylcholine to a basal level of less than 5%. nte1Delta cells grown in the absence of inositol and in the presence of choline exhibited lower levels of dimyristoylphosphatidylcholine than wild type cells grown under these same conditions, but these levels remained largely constant after the addition of inositol. These results are discussed in relationship to transcriptional regulation known to be linked to lipid metabolism in yeast.