Increased contractile activity as induced by chronic low-frequency stimulation evoked in rat fast-twitch muscle an almost immediate increase in the ratio between structure-bound and free hexokinase. In addition, an up to 14-fold rise in total hexokinase activity occurred after two weeks of stimulation indicating that glucose phosphorylation became a limiting step of glucose utilization under these conditions. The increase in hexokinase activity was transitory as prolonged stimulation led to a leveling off and steep decline with an apparent half-life of 2.5 days after three weeks of stimulation. The transient increase in glucose phosphorylating capacity can be explained by previous observations indicating that prolonged stimulation leads to a shift from a carbohydrate-based to a fatty-acid-based energy metabolism. Using an isozyme-specific sandwich ELISA, it was shown that both increases and decreases in total hexokinase activity were matched by corresponding changes in the amount of hexokinase isozyme II protein. Increases in both total hexokinase activity (3-4-fold) and hexokinase II protein content were also observed after denervation in rat fast-twitch muscle. In view of reports in the literature, it is suggested that the elevations in hexokinase II observed with increased contractile activity and denervation relate to enhanced glucose uptake and utilization.