It has been shown that a significant number of growth factors transduce intracellular signals via the same pathway which is induced by oncogene products. Recently, it has been reported that insulin receptor associates with PI3-kinase and activates production of the D-3 phosphoinositides. We studied the influence of insulin on PI 3-kinase activity in the isolated rat adipocyte. Insulin treatment of adipocytes caused a remarkable increase in the PI kinase activity in immunoprecipitates induced by anti-phosphotyrosine antibody. PI kinase activity was half-maximal at 1.15×10-8M of insulin. Enzyme activity in the immunoprecipitates from insulin-stimulated adipocytes was capable of phosphorylating phosphatidylinositol, phosphatidyl-inositol4-phosphate and phosphatidyl-inositol 4, 5-bisphosphate. A deacylated product of phosphorylated phosphatidyl-inositol corresponding to phosphatidylinositol 3-phosphate, was eluted on anion exchange HPLC at 34 min. This demonstrates that insulin regulates PI 3-kinase activity not only in cultured cell lines, but also in isolated rat adipocytes.