Previous studies have shown that catalytic function is associated with both halves of the Type II isozyme of mammalian hexokinase, while the Type I isozyme is functionally differentiated into a catalytic C-terminal half and regulatory N-terminal half. The Type III isozyme has now been shown to be similar to the Type I isozyme in its functional organization. Chimeras composed of the N-terminal half of Type III hexokinase and the C-terminal half of either Type I or Type II hexokinase have activities that can be attributed to the C-terminal half and are similar in activity to chimeras composed of the C-terminal half of Type III and the intrinsically inactive N-terminal domain of Type I or the inactivated (by site-directed mutation) N-terminal half of Type II hexokinase. Virtually no activity was seen with chimeras constructed with the N-terminal half of the Type III isozyme and catalytically inactive (by site-directed mutation) C-terminal halves of Type I or Type II hexokinase. Substrate inhibition by Glc is seen only with the Type III isozyme and with chimeric forms containing the C-terminal half of Type III hexokinase and the N-terminal half of Type I or Type II isozyme, the latter inactivated by site-directed mutation; this is attributed to conformational changes induced by binding of Glc to a low affinity site in the N-terminal half, with subsequent effect on catalytic activity of the C-terminal half. These results also provide further insight into the role of interactions (or lack of interactions) between the N- and C-terminal halves in the inhibition of the Type I-III isozymes by Glc-6-P, its antagonism by low concentrations of Pi, and the inhibition seen at higher concentrations of Pi.