Type II diacylglycerol kinase (DGK) isozymes (␦, , and ) have a pleckstrin homology domain (PH) at their N termini. Here, we investigated the lipid binding properties of the PHs of type II DGK isozymes using protein-lipid overlay and liposome binding assays. The PH of DGK showed the most pronounced binding activity to phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P 2 ) among the various glycero-and sphingolipids including PI 3,4,5-trisphosphate, PI 3,4-bisphosphate, PI 3-phosphate, PI 4-phosphate, and PI 5-phosphate. Moreover, the PI(4,5)P 2 binding activity of the DGK-PH was significantly stronger than that of other type II DGK isozymes. Notably, compared with the PH of phospholipase C (PLC) ␦1, which is generally utilized as a cellular PI(4,5)P 2 -probe, the DGK-PH is equal to or superior than the PLC␦1-PH in terms of affinity and selectivity for PI(4,5)P 2 . Furthermore, in COS-7 cells, GFP-fused wild-type DGK1 and its PH partly translocated from the cytoplasm to the plasma membrane where the PLC␦1-PH was co-localized in response to hyperosmotic stress in an inositol 5-phosphatase-sensitive manner, whereas a PH deletion mutant did not. Moreover, K74A and R85A mutants of DGK-PH, which lack the conserved basic amino acids thought to ligate PI(4,5)P 2 , were indeed unable to bind to PI(4,5)P 2 and co-localize with the PLC␦1-PH even in osmotically shocked cells. Overexpression of wild-type DGK1 enhanced EGF-dependent phosphorylation of ERK, whereas either K74A or R85A mutant did not. Taken together, these results indicate that the DGK-PH preferentially interacts with PI(4,5)P 2 and has crucial roles in regulating the subcellular localization and physiological function of DGK. Moreover, the DGK-PH could serve as an excellent cellular sensor for PI(4,5)P 2 . Diacylglycerol kinase (DGK)2 phosphorylates diacylglycerol to produce phosphatidic acid (1-5). Diacylglycerol (6 -8) and phosphatidic acid (PA) (9, 10) are well recognized as lipid second messengers. DGK appears to participate in various physiological events by modulating the balance between the two bioactive lipids diacylglycerol and phosphatidic acid (1-5). Ten mammalian DGK isozymes (␣, ␤, ␥, ␦, ⑀, , , , , and ) containing two or three characteristic zinc finger-like C1 domains and a common catalytic region are divided into five groups (types I-V) according to their structural features (1-5).Type II DGKs (11) consist of DGK␦ (12), - (13), and -(14). Moreover, alternative splicing products of DGK␦ (␦1 and ␦2) (15) and -(1 and 2) (16) have been identified. Type II DGKs have a pleckstrin homology domain (PH) in common at their N termini and a catalytic domain that is divided into two subdomains (catalytic subdomains a and b). DGKs ␦1, ␦2, and 2, but not DGKs 1 and , contain a sterile ␣-motif domain at their C termini. It has been demonstrated that DGKs ␦1, ␦2, and 2 form oligomers through interactions between their sterile ␣-motif domains and that this oligomer formation regulates the activities and subcellular localization of these DGK isoforms (15)(16)(1...