Several glycolytic enzymes exist in muscle as free and structure-bound forms. A fraction of hexokinase (HK) is associated with the outer mitochondrial membrane. Phosphofructokinase (PFK) and aldolase (ALD) bind to F-actin, and AMP deaminase (AMPase) interacts with myosin. Using low-frequency stimulation (10 Hz, 24 h/d), we studied in rat fast-twitch muscle effects of contractile activity on soluble and structure-bound forms of these enzymes. Phosphoglucose isomerase (PGI), a soluble enzyme, was also examined. Fractional extraction was applied to study the intracellular distribution of soluble and bound enzyme activities 5 min, 1 h, 3 h, 1 d, and 7 d after the onset of stimulation. Confirming previous findings, total HK activity increased 7-fold in 7-d-stimulated muscles, whereas PFK, ALD, and PGI were reduced, ranging between 55% and 80% of their normal activities. AMPase activity was unaltered. At the time points studied, no changes were found in the extraction behavior of PGI and AMPase. The fraction of bound ALD increased slightly (12%). However, the distribution of HK and PFK was markedly altered. Bound PFK increased from 50% in the control to 85% in 7-d-stimulated muscles. Bound HK rose from 52% to 83% during the same time period. The increase in PFK binding was steep and occurred mainly within the first minutes and hours. The increase in HK binding occurred with some delay, but was significant in muscles stimulated for more than 1 h. In view of the altered kinetic properties of F-actin-bound PFK (alleviated allosteric inhibition by ATP) and bound HK (elevated catalytic activity), these changes are interpreted as early responses to match the metabolic demands during maximal contractile activity imposed on a muscle not programmed for sustained activity: Enhanced binding of PFK serves to accelerate glycolytic flux immediately after the onset of stimulation, whereas mitochondrial binding of HK facilitates the phosphorylation of exogenous glucose when glycogen stores have been depleted.