It is well-established that the endoplasmic reticulum is the major site of phosphatidylinositol (PtdIns) synthesis. The PtdIns synthetic ability of other organelles, such as plasma membrane and nucleus, remains controversial. In the present study, we re-examine this question by comparing PtdIns synthesis in isolated cytoplasts (enucleated cells) with that in corresponding karyoplasts (nuclei surrounded by plasma membrane but lacking most cytoplasmic components). We report that cytoplasts are competent to carry out both basal and stimulated PtdIns synthesis as well as polyphosphoinositide hydrolysis, while karyoplasts can neither synthesize PtdIns nor hydrolyze phosphoinositides in response to agonists. The karyoplasts are, however, capable of synthesizing phosphatidylcholine (PtdCho), as previously reported. From these data, we conclude that PtdIns synthesis is limited to cytoplasmic components, and cannot be sustained by either plasma membrane or nucleus under conditions that permit robust PtdCho synthesis. (c) 2006 Elsevier Inc. All rights reserved.