The addition of naturally occurring polyamines and inorganic ions to an in vitro protein-synthesizing system improved the extent and fidelity of translation. In such an optimized system, regeneration of the nucleoside triphosphates with phosphoenolpyruvate and pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) reduced further the missense error frequency to the in vivo level as well as enhanced the extent of translation. The effect of nucleoside triphosphate regeneration was shown to be due primarily to the increase in the ratio of adenosine and guanosine triphosphates to their hydrolysis products and only marginally due to the increase in the absolute concentrations of the nucleoside triphosphates.