Phosphatidylcholine (PtdCho) is the major phospholipid component of eukaryotic membranes and deciphering the molecular mechanisms regulating PtdCho homeostasis is necessary to fully understand many pathophysiological situations where PtdCho metabolism is altered. This concept is illustrated in this review by summarizing recent evidence on Nte1p, a yeast endoplasmic reticulum resident phospholipase B that deacylates PtdCho producing intracellular glycerophosphocholine. The mammalian and Drosophila homologues, neuropathy target esterase and swiss cheese, respectively, have been implicated in normal brain development with increased intracytoplasmic vesicularization and multilayered membrane stacks as cytological signatures of their absence. Consistent with a role in lipid and membrane homeostasis, Nte1p-mediated PtdCho deacylation is strongly affected by Sec14p, a component of the yeast secretory machinery characterized by its ability to interface between lipid metabolism and vesicular trafficking. The preference of Nte1p toward PtdCho produced through the CDP-choline pathway and the downstream production of choline by the Gde1p glycerophosphodiesterase for resynthesis of PtdCho by the CDP-choline pathway are also highlighted.