Nucleoside diphosphate (NDP) kinase is a ubiquitous enzyme that has been described to have regulatory functions. In addition to its classical enzymatic activity, NDP kinases have been characterized as inhibitors of metastasis, as a factor stimulating gene transcription, and as a protein kinase. In this report we show some characteristics of the autophosphorylation of homogeneous NDP kinase and make a comparison with that of other proteins in crude extracts. By using labeled substrates and fluorescence quenching analysis, we prove that Mg2+ is indeed necessary for the two steps of the ping-pong reaction to take place and present evidence that NTPs or NDPs, when uncomplexed to divalent cations, may not bind the active site in a comparable way to NTP . Mg2+ and NDP . Mg2+. However, even extremely small concentrations of Mg2+ suffice for maximal autophosphorylation which is obtained with Mg2+ in the nanomolar range and 100 microM ATP using homogeneous enzyme. Moreover, lower autophosphorylation levels were observed with increasing concentrations of Mg2+. The autophosphorylation equilibrium varied from 0.19 to 1.6 upon the inclusion of 10 mM EDTA to produce low Mg2+ concentrations. Under optimal conditions (low Mg2+ concentrations and short incubation times) NDP kinase was the only protein phosphorylated in crude extracts from Candida albicans, indicating that the autophosphorylation properties of the enzyme are very singular.