The aim of this study was to investigate the effects of an enzymatic removal of glycogen on excitation-contraction coupling in mechanically skinned fibres of rat fast-twitch muscles, with a focus on the changes in the function of Na+-K+-pump and ryanodine receptor (RyR). Glycogen present in the skinned fibres and binding to microsomes was removed using glucoamylase (GA). Exposure of whole muscle to 20 U mL-1 GA for 6 min resulted in a 72% decrease in the glycogen content. Six minutes of GA treatment led to an 18 and a 22% reduction in depolarization- and action potential-induced forces in the skinned fibres, respectively. There was a minor but statistically significant increase in the repriming period, most likely because of an impairment of the Na+-K+-pump function. GA treatment exerted no effect on the maximum Ca2+ release rate from the RyR in the microsomes and the myofibrillar Ca2+ sensitivity in the skinned fibres. These results indicate that reduced glycogen per se can decrease muscle performance due to the impairment of SR Ca2+ release and suggest that although Na+-K+-pump function is adversely affected by reduced glycogen, the extent of the impairment is not sufficient to reduce Ca2+ release from the sarcoplasmic reticulum. This study provides direct evidence that glycogen above a certain amount is required for the preservation of the functional events preceding Ca2+ release from the sarcoplasmic reticulum.