Using a teased muscle fiber preparation, we determined the activity of mitochondrially bound hexokinase in rat fast-twitch muscle under control conditions and after low-frequency stimulation periods for up to 2 h. As compared to soluble hexokinase, mitochondrial binding led to stimulation of glucose 6-phosphate production. Low-frequency stimulation greatly enhanced glucose 6-phosphate formation which was 100% and 250% elevated after 1 and 2 h, respectively. These observations point to a mechanism which rapidly increases the catalytic activity of hexokinase through binding to the mitochondrial surface.