Using phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] prepared from phosphatidylinositol 4,5-bisphosphate and inositolphospholipid 3-kinase, we identified in bovine thymus extracts the enzyme activity which catalyzed dephosphorylation of PtdIns(3,4,5)P3, to produce phosphatidylinositol biphosphate. Since bovine thymus exhibited the highest level of activity among tissues screened, we tried to purify this enzyme PtdINs(3,4,5)P3 phosphatase from bovine thymus. After sequential chromatographies using S-Sepharose, heparin-Sepharose, blue Sepharose, and Toyopearl HW55, the enzyme was purified 1875-fold with a yield of 10%. SDS/PAGE analysis revealed that a 120-kDA protein band copurified with the enzyme activity. The apparent molecular mass of the active protein was 120 kDa on size-exclusion chromatography, suggesting that the 120-kDa band on SDS/PAGE is the PtdIns(3,4,5)P3 phosphatase. Since PtdIns(3,4,5)P3 phosphatase seemed to be the only activity that metabolized PtdIns(3,4,5)P3, and the enzyme did not hydrolyze phosphatidylinositol 4,5-biphosphate, the enzyme may play a critical role in the inositolphospholipid 3-kinase signalling.