This study examined the impact of acute high-intensity exercise on the rate of Ca²⁺uptake and release and Ca²⁺-stimulated adenosine triphosphatase (ATPase) activity of the sarcoplasmic reticulum (SR) in the soleus muscle (SOL) and the superficial region of the vastus lateralis muscle (VS) of rats. The animals were run on a 10% grade at 50 m/min of a motorized treadmill until fatigued (The average time to exhaustion was 306 sec.) . At exhaustion, glycogen concentrations were 65% and 85% less in the SOL and VS, respectively. The rate of Ca²⁺release induced by 4-chloro-m-cresol was un-changed in fatigued SOL and VS. The rate of Ca²⁺uptake stimulated by adenosine triphosphate (ATP) was significantly lower following exercise in VS but not in SOL. This lower rate observed in VS was paralleled by decreased catalytic activity of SR Ca²⁺-ATPase. The rate of Ca2+ uptake measured using adenosine diphosphate and phosphocreatine, as substrate was lower than that of ATP in fatigued VS. These findings suggest that, in fast-twitch muscles, high-intensity exercise not only reduces SR Ca²⁺-ATPase activity but also elicits a decrease in creatine kinase activity, probably resulting from nitric oxide that is produced during exercise.