Products of phosphatidylinositol (PI) turnover have recently been implicated as regulators of cell growth and differentiation. Transformation of cells in culture by infection with certain viruses (Rous sarcoma virus, Kirsten sarcoma virus, and polyoma virus) or by transfection with the oncogenes carried by these viruses affect the steady-state level of intermediates in the PI turnover pathway. In addition, immunoprecipitates of the transforming gene products of Rous sarcoma virus and polyoma virus contain activities of certain enzymes in the PI turnover pathway. We have previously reported that polyoma middle T immunoprecipitates can catalyze phosphorylation of PI to phosphatidylinositol-4-phosphate (PIP). This activity is not intrinsic to middle T or pp60c-src but is due to a cellular enzyme that specifically associates with the middle T/pp60c-src complex. The PI kinase is found in immunoprecipitates of the middle t protein from polyoma viruses that are capable of cell transformation but does not associate with mutants of middle t defective in transformation, suggesting that this association may be important for transformation. Two PI kinases from fibroblasts (type I and type II) that are separable by anion exchange chromatography have been partially purified and characterized. These enzymes differ in their Km for ATP as well as their Ki for adenosine and ADP. Only the type I PI kinase specifically associates with the transformation-competent mutants of middle T.