Phosphatidylinositol kinase present in the membranes of Xenopus laevis oocytes was characterized. 2. The enzyme requires Mg2+ or Mn2+ at 10 mM and exogenous phosphatidylinositol (50 microM) increases the formation of phosphatidylinositol-4-phosphate. 3. The oocyte phosphatidylinositol kinase cannot use GTP as a phosphate donor but this compound inhibits competitively the utilization of ATP. 4. Addition of phosphatidylserine and phosphatidylinositol-4,5-bisphosphate stimulates the phosphorylation of phosphatidylinositol but 2,3-bisphosphoglycerate at 5 mM concentration is a strong inhibitor of the reaction.