This chapter describes the best methods available for regeneration of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and adenosine monophosphate (AMP) and details several applications of these methods in enzyme-catalyzed syntheses. In particular, it give procedures for synthesis of three phosphate donors—acetyl phosphate, phosphoenolpyruvate, and methoxycarbonyl phosphates—and apply these reagents to syntheses of sn-glycerol 3-phosphate, dihydroxyacetone phosphate, glucose 6-phosphate, 5-phospho-α-D-ribosyl pyrophosphate, and uridine-5'-diphosphoglucose. Three procedures for the enzymatic regeneration of ATP are presently available, which are useful in practical-scale organic synthesis. One is based on acetyl phosphate (AcP) as the phosphorylating agent and acetate kinase as the catalyst; the second uses phosphoenolpyruvate (PEP) and pyruvate kinase; and the third uses methoxycarbonyl phosphate [CH3OC(O)OPO3 2-, MCP] and acetate kinase. The advantages and disadvantages of each method are summarized in the chapter. Both pyruvate kinase and acetate kinase have high specific activity and show excellent stability in immobilized form. Pyruvate kinase is currently the less expensive enzyme. Further, it is effective for regeneration of ATP from ADP at lower concentrations of ADP than is acetate kinase, because the Michaelis constant for pyruvate kinase is lower than that for acetate kinase. In practice, for most synthetic applications, either acetyl phosphate/acetate kinase or phosphoenolpyruvate/pyruvate kinase is used for the regeneration of ATP.