The preference of maize (Zea mays L.) mitochondrial hexokinase (EC 2.7.1.1.) for glucose and fructose and the ADP regulation were evaluated. The Michaelis-Menten constants (Km) varied between 0.02 and 0.09 mM for glucose and from 2 to 6 mM for fructose as substrates. The value of Vmax was five times higher in the presence of glucose as compared with fructose in membrane-bound enzyme preparations. It was shown that ADP produced from the reaction inhibits the hexokinase activity (Ki=20–50 μM). However, the inhibition was very specific for adenine nucleotide. Only a small inhibition was observed when 1 mM of UDP, CDP or GDP was included in the assay medium. Nevertheless, the ADP inhibition was observed only when glucose was phosphorylated. In assay conditions where fructose serves as substrate, the affinity for ADP decreased by 10-fold (Ki varied between 500 and 1 000 μM). These kinetics properties were also observed in partially purified soluble enzyme preparations. These data suggest that the type of hexose bound to the catalytic site modulates the ADP control of maize mitochondrial hexokinase.