Here, we identify a novel rat phosphatidylinositol-5-phosphate 4-kinase, phosphatidylinositol-phosphate kinase IIgamma (PIPKIIgamma). PIPKIIgamma comprises 420 amino acids with a molecular mass of 47,048 Da, showing greater homology to the type IIalpha and IIbeta isoforms (61.1 and 63.7% amino acid identities, respectively) of phosphatidylinositol-phosphate kinase than to the type I isoforms. It is predominantly expressed in kidney, with low expression in almost all other tissues. PIPKIIgamma was found to have phosphatidylinositol-5-phosphate 4-kinase activity as demonstrated in other type II kinases such as PIPKIIalpha. The PIPKIIgamma that is present endogenously in rat fibroblasts, PC12 cells, and rat whole brain lysate or that is exogenously overexpressed in COS-7 cells shows a doublet migrating pattern on SDS-polyacrylamide gel electrophoresis. Alkaline phosphatase treatment and metabolic labeling in [32P]orthophosphate experiments revealed that PIPKIIgamma is phosphorylated in vivo, resulting in a shift in its electrophoretic mobility. Phosphorylation is induced by treatment of mitogens such as serum and epidermal growth factor. Immunostaining experiments and subcellular fractionation revealed that PIPKIIgamma localizes dominantly in the endoplasmic reticulum (ER). Phosphorylation also occurs in the ER. Thus, PIPKIIgamma may have an important role in the synthesis of phosphatidylinositol bisphosphate in the ER.