The first step in metabolism of glucose (Glc) is usually phosphorylation, catalyzed by hexokinase. However, the Glc-6-P produced can then enter one or more of several alternative pathways. Selective expression of isozymic forms of hexokinase, differing in catalytic and regulatory properties as well as subcellular localization, is likely to be an important factor in determining the pattern of Glc metabolism in mammalian tissues/cells. Despite their overall structural similarity, the Type I, Type II and Type III isozymes differ in important respects. All three isozymes are inhibited by the product, Glc-6-P, but with the Type I isozyme, this inhibition is antagonized by P(I), whereas with the Type II and Type III isozymes, P(i) actually causes additional inhibition. Reciprocal changes in intracellular levels of Glc-6-P and P(i) are closely associated with cellular energy status, and it is proposed that the response of the Type I isozyme to these effectors adapts it for catabolic function, introducing Glc into glycolytic metabolism for energy production. In contrast, the Type II, and probably the Type III, isozymes are suggested to serve primarily anabolic functions, e.g. to provide Glc-6-P for glycogen synthesis or metabolism via the pentose phosphate pathway for lipid synthesis. Type I hexokinase binds to mitochondria through interaction with porin, the protein that forms channels through which metabolites traverse the outer mitochondrial membrane. Several experimental approaches have led to the conclusion that the Type I isozyme, bound to actively phosphorylating mitochondria, selectively uses intramitochondrial ATP as substrate. Such interactions are thought to facilitate coordination of the introduction of Glc into glycolysis, via the hexokinase reaction, with the terminal oxidative stages of Glc metabolism occurring in the mitochondria, thus ensuring an overall rate of Glc metabolism commensurate with cellular energy demands and avoiding excessive production of lactate. The Type II isozyme also binds to mitochondria. Whether such coupling occurs with mitochondrially bound Type II hexokinase in normal tissues, and how it might be related to the proposed anabolic role of this isozyme, remain to be determined. The Type III isozyme lacks the hydrophobic N-terminal sequence known to be critical for binding of the Type I and Type II isozymes to mitochondria. Immunolocalization studies have indicated that, in many cell types, the Type III has a perinuclear localization, the possible metabolic consequences of which remain unclear.