Brain hexokinase (HKI) is inhibited potently by its product glucose 6-phosphate (G6P); however, the mechanism of inhibition is unsettled. Two hypotheses have been proposed to account for product inhibition of HKI. In one, G6P binds to the active site (the C-terminal half of HKI) and competes directly with ATP, whereas in the alternative suggestion the inhibitor binds to an allosteric site (the N-terminal half of HKI), which indirectly displaces ATP from the active site. Single mutations within G6P binding pockets, as defined by crystal structures, at either the N- or C-terminal half of HKI have no significant effect on G6P inhibition. On the other hand, the corresponding mutations eliminate product inhibition in a truncated form of HKI, consisting only of the C-terminal half of the enzyme. Only through combined mutations at the active and allosteric sites, using residues for which single mutations had little effect, was product inhibition eliminated in HKI. Evidently, potent inhibition of HKI by G6P can occur from both active and allosteric binding sites. Furthermore, kinetic data reported here, in conjunction with published equilibrium binding data, are consistent with inhibitory sites of comparable affinity linked by a mechanism of negative cooperativity.