A nucleosidediphosphate kinase (NDP-kinase) has been highly purified from E. coli. The purified enzyme was found to have a molecular weight of approximately 64,000 daltons and be a 16,000 dalton polypeptide tetramer. The enzyme rapidly formed a phosphoenzyme when incubated with ATP in the presence of divalent cations such as Mg2+ and Ca2+ (1 mM) even at low temperatures (below 4 degrees C). The available evidence suggests that the phosphoenzyme is an enzyme-bound high energy phosphate intermediate, which functions as an intermediary in NDP-kinase action.