Kinetic studies were used to investigate the mode of brain hexokinase (EC 2.7.1.1, ATP:D-hexose 6-phosphotransferase) regulation by glucose 6-phosphate (glucose-6-P), ADP, and inorganic phosphate (Pi). A model for regulation of brain hexokinase by glucose-6-P and Pi had been proposed from initial-rate studies and binding experiments [Ellison, W. R., Lueck, J. D., & Fromm, H. J. (1975) J. Biol. Chem. 250, 1864-1871]. The results of the present investigation demonstrate that Pi is an activator of the brain hexokinase reaction when the reaction is studied in the nonphysiological direction. Evidence is presented which indicates that the back-reaction substrates and Pi can bind the enzyme simultaneously, and the suggestion is made that Pi binds to an allosteric site on the enzyme. These findings are in marked contrast to results obtained in the absence of ADP which convincingly demonstrate that glucose-6-P and Pi are mutually exclusive binding ligands for brain hexokinase. The kinetic data can be reconciled with the model for hexokinase regulation within the context of the well-established kinetic mechanism for brain hexokinase.