In an E. coli cell-free protein synthesis system, the addition of an inorganic polyphosphate [poly(P)] with polyphosphate:AMP phosphotransferase (PAP), which regenerates AMP to ADP, increased the amount of protein synthesis. The maximum yield of the translation product (green fluorescent protein) in the E. coli cell-free system provided by Roche Diagnostics (RTS-100) was 1.16 mg/ml under the optimum reaction condition, which corresponded to a 5.7-fold of that obtained under the standard reaction condition described in the manufacturer's protocol. Interestingly, poly(P) alone enhanced protein synthesis to some extent. When we added poly(P) to the reaction mixture, ATP was consumed at a faster rate, leading to a rapid accumulation of AMP. By adding both poly(P) and PAP to the reaction mixture, an efficient ATP regeneration reaction derived from AMP occurred and the ATP level was recovered. Since the protein synthesis enhancement by poly(P) was also observed when mRNA was added as the template in the reaction, poly(P) accelerated the translation reaction by directly affecting the translation machinery. This also occurred when we used the Pure-system Classic Mini kit (Post Genome Institute) that contained the minimum requirements (pure enzymes and chemicals) for translation and transcription. We also observed that poly(P) extended the half-life of the mRNA template.