The control of glycolysis in contracting muscle is not fully understood. The aim of the present study was to examine whether activation of glycolysis is mediated by factors related to the energy state or by a direct effect of Ca2+ on the regulating enzymes. Extensor digitorum longus muscles from rat were isolated, treated with cyanide to inhibit aerobic ATP production and stimulated (0.2 s trains every 4 s) until force was reduced to 70% of initial force (control muscle, referred to as Con). Muscles treated with BTS (N-benzyl-p-toluene sulfonamide), an inhibitor of cross-bridge cycling without affecting Ca2+ transients, were stimulated for an equal time period as Con. Energy utilization by the contractile apparatus (estimated from the observed relation between ATP utilization and force-time integral) was 60% of total. In BTS, the force-time integral and ATP utilization were only 38 and 58% of those in Con respectively. Glycolytic rate in BTS was only 51% of that in Con but the relative contribution of ATP derived from PCr (phosphocreatine) and glycolysis and the relation between muscle contents of PCr and Lac (lactate) were not different. Prolonged cyanide incubation of quiescent muscle (low Ca2+) did not change the relation between PCr and Lac. The reduced glycolytic rate in BTS despite maintained Ca2+ transients, and the unchanged PCr/Lac relation in the absence of Ca2+ transients, demonstrates that Ca2+ is not the main trigger of glycogenolysis. Instead the preserved relative contribution of energy delivered from PCr and glycolysis during both conditions suggests that the glycolytic rate is controlled by factors related to energy state.