Hexokinase isoform I binds to mitochondria of many cell types. It has been hypothesized that this association is regulated by changes in the concentrations of specific cellular metabolites. To study the distribution of hexokinase in living cells, fluorophore-labeled functional hexokinase I was prepared. After microinjection into A7r5 smooth muscle cells, hexokinase localized to distinct structures identified as mitochondria. The endogenous hexokinase demonstrated a similar distribution with the use of immunocytochemistry. 2-Deoxyglucose elicited an increase in glucose 6-phosphate (G-6-P) and a decrease in ATP levels and diminished hexokinase binding to mitochondria in single cells. 3-O-methylglucose elicited slowly developing decreases in all three parameters. In contrast, cyanide elicited a rapid decrease in both ATP and hexokinase binding. Analyses of changes in metabolite levels and hexokinase binding indicate a positive correlation between binding and cell energy state as monitored by ATP. On the other hand, only in the presence of 2-deoxyglucose was the predicted inverse correlation between binding and G-6-P observed. Unlike the relatively large changes in distribution observed with the fluorescent-injected hexokinase, cyanide caused only a small decrease in the localization of endogenous hexokinase with mitochondria. These findings suggest that changes in the concentrations of specific metabolites can alter the binding of hexokinase I to specific sites on mitochondria. Moreover, the apparent difference in sensitivity of injected and endogenous hexokinase to changes in metabolites may reflect the presence of at least two classes of binding mechanisms for hexokinase, with differential sensitivity to metabolites.