It was recently reported that hyperglycemia provokes a rapid and sustained translocation of glucokinase in rat pancreatic B-cells, and it was speculated that this may be associated with enhancement of its catalytic activity, as possibly attributable to the mitochondrial binding of the enzyme. In the present work, the activities of both hexokinase and glucokinase were measured in particulated and cytosolic subcellular fractions prepared from islets first incubated for 60 min either in the absence of exogenous nutrient or in the presence of D-glucose, tested at both low (2.8 mmol/L) and high (16.7 mmol/L) concentrations. The relative contribution of the cytosolic domain to the total activity of glucokinase recovered in the two subcellular fractions was higher in islets deprived of exogenous nutrient than in islets first exposed to 2.8 or 16.7 mmol/L D-glucose, the results obtained at each of the latter two hexose concentrations being comparable to one another. The subcellular distribution of hexokinase, however, was not significantly different in islets deprived of D-glucose or exposed to the hexose. These findings are interpreted as indicative of an energy-dependent translocation of glucokinase in the B-cell, distinct from the redistribution of the enzyme occurring in response to a rise in D-glucose concentration above its physiological value.