The synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], the immediate precursor of intracellular signals generated by calcium-mobilizing hormones and growth factors, is initiated by the conversion of phosphatidylinositol to phosphatidylinositol 4-phosphate [PtdIns(4)P] by phosphatidylinositol 4-kinase (PtdIns 4-kinase). Although cells contain several PtdIns 4-kinases, the enzyme responsible for regulating the synthesis of hormone-sensitive PtdIns(4,5)P2 pools has not been identified. In this report we describe the inhibitory effect of micromolar concentrations of wortmannin (WT) on the synthesis of hormone-sensitive PtdIns(4)P and PtdIns(4,5)P2 pools in intact adrenal glomerulosa cells, and the presence of a WT-sensitive PtdIns 4-kinase in adrenocortical extracts. In addition to its sensitivity to the PtdIns 3-kinase inhibitor WT, this enzyme is distinguished from the recognized membrane-bound PtdIns 4-kinases by its molecular size and weak membrane association. Inhibition of this PtdIns 4-kinase by WT results in rapid loss of the hormone-sensitive PtdIns(4,5)P2 pool in angiotensin II-stimulated glomerulosa cells. Consequently, WT treatment inhibits the sustained but not the initial increases in inositol 1,4,5-trisphosphate and cytoplasmic [Ca2+] in a variety of agonist-stimulated cells, including adrenal glomerulosa cells, NIH 3T3 fibroblasts, and Jurkat lymphoblasts. These results indicate that a specific WT-sensitive PtdIns 4-kinase is critical for the maintenance of the agonist-sensitive polyphosphoinositide pool in several cell types.