Type I phosphatidylinositol 4-phosphate (PtdIns(4)P) 5-kinases (PIP5K) catalyze the synthesis of phosphatidylinositol 4,5-bisphosphate, an essential lipid molecule in various cellular processes. Here, we report the cloning of the third member (PIP5K␥) and the characterization of members of the type I PIP5K family. Type I PIP5K␥ has two alternative splicing forms, migrating at 87 and 90 kDa on SDS-polyacrylamide gel electrophoresis. The amino acid sequence of the central portion of this isoform shows approximately 80% identity with those of the ␣ and  isoforms. Northern blot analysis revealed that the ␥ isoform is highly expressed in the brain, lung, and kidneys. Among three isoforms, the  isoform has the greatest V max value for the PtdIns(4)P kinase activity and the ␥ isoform is most markedly stimulated by phosphatidic acid. By analyzing deletion mutants of the three isoforms, the minimal kinase core sequence of these isoforms were determined as an approximately 380-amino acid region. In addition, carboxyl-terminal regions of the  and ␥ isoforms were found to confer the greatest V max value and the highest phosphatidic acid sensitivity, respectively. It was also discovered that lysine 138 in the putative ATP binding motif of the ␣ isoform is essential for the PtdIns(4)P kinase activity. As was the case with the ␣ isoform reported previously (Shibasaki, Y., Ishihara, H., Kizuki, N., Asano, T., Oka, Y., Yazaki, Y. (1997) J. Biol. Chem. 272, 7578 -7581), overexpression of either the  or the ␥ isoform induced an increase in short actin fibers and a decrease in actin stress fibers in COS7 cells. Surprisingly, a kinase-deficient substitution mutant also induced an abnormal actin polymerization, suggesting a role of PIP5Ks via structural interactions with other molecules.Recent advances in cell biology have revealed that phosphoinositide metabolism plays an essential role in various cellular processes. Synthesis and breakdown of certain phosphoinositides at appropriate times and intracellular sites appear to be required for complex regulation of these cellular processes. One of the phosphoinositides, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ), 1 is located at an important branchpoint in phosphoinositide metabolism. PtdIns(4,5)P 2 serves as a substrate for phosphoinositide-specific phospholipase C (EC 3