Hexokinase PII is not inhibited by high Mg-ATP concentrations if the Mg2+-free ATP is kept at low levels (0.01 mM) in the assay mixture. Hexokinase PI activity is not affected either by Mg2+-free ATP nor by free Mg2+ in the assay mixture. Thus, hexokinase PI and PII activities appear not to be regulated by substrate inhibition as proposed previously [Kopetzki, E. & Entian, K. D. (1985) Eur. J. Biochem. 146, 657-662]. However, the level of Mg2+-free ATP in the hexokinase PII assay mixture strongly affects the enzyme activity by decreasing the Vmax and increasing the Km value for Mg-ATP from 0.15 mM to 5.0 mM. The physiological role of this inhibition, which has not been described previously, was investigated by determining the cytosolic ATP and Mg2+ concentrations in yeast cells grown under derepressing and repressing conditions. Derepression is accompanied by an important loss of Mg2+ from the cells, maintaining the ATP concentration constant. This produces an increase of Mg2+-free ATP in the cytosol from 0.01 mM to 0.1 mM. This free ATP concentration would lead to a maximal inhibition of hexokinase PII.