In order to elucidate the location of chorismate mutase and prephenate dehydrogenase on E.coli T-protein, protein limited digestion and fragmentation cloning were employed. Fragment 1～93 and fragment 96～373 were cloned and expressed according to the results of limited digestion of T-protein respectively. Two fragments were found to have chorismate mutase and prephenate dehydrogenase activities respectively. In a conclusion, E.coli T-protein do have independent domains, N-terminal 93 amino acids belongs to chorismate mutase and C-terminal 277 amino acids belongs to prephenate dehydrogenase.