A cell-free extract of Escherichia coli, even after exhaustive dialysis, was capable of phosphorylating 5'-AMP to ADP and ATP. When cyclic AMP phosphodiesterase was present in the extract, the conversion of cyclic AMP to ADP and ATP occurred. If 3H or 32P-labeled 5'-AMP or cyclic AMP was added to the extract, a significant portion of the radioactivity was incorporated into the 3'-terminus of tRNA species. The phosphorylating capacity was sedimentable after centrifuging at 100,000 g for 3 hours. The pellet contained phosphate polymers which are neither DNA, RNA or proteins, while the supernate contained little of this material. The addition of authentic inorganic polyphosphates to the supernate restored the original phosphorylating capacity of the extract. Thus, it is concluded that the phosphorylating property is partly due to inorganic polyphosphates. A method for assaying total inorganic polyphosphates has been developed.