Accumulating evidence suggests that phosphatidylinositol metabolism is essential for membrane traffic in the cell. Of particular importance, phosphatidylinositol transfer protein and the type I phosphatidylinositol-4- phosphate 5-kinase (PI4P5K) have been identified as cytosolic components required for ATP-dependent, Ca2+-activated secretion. In order to identify PI4P5K isoforms that may play important roles in regulated insulin secretion from pancreatic β-cells, we employed the polymerase chain reaction with degenerate primers and screening of a cDNA library of the murine pancreatic β-cell line MIN6. Two novel cDNAs, designated PI4P5K-Iα and PI4P5K-Iβ, were identified, which contained complete coding sequences encoding 539- or 546-amino acid proteins, respectively. These cDNAs were expressed in mammalian cells with an adenoviral expression vector. Proteins of both isoforms migrated at 68 kDa on SDS-polyacrylamide gel electrophoresis and exhibited phosphatidylinositol-4-phosphate 5-kinase activity, which was activated by phosphatidic acid, indicating that these proteins were type I isoforms. While these isoforms share a marked amino acid sequence homology in their central portion, the amino- and carboxyl-terminal regions differ significantly. Northern blot analysis depicted that tissue distributions differed between the two isoforms. Molecular identification of type I PI4P5K isoforms in insulin-secreting cells should provide insights into the role of phosphatidylinositol metabolism in regulated exocytosis of insulin-containing large dense core vesicles.