Miss-function of mitochondria and an impairment of the excitation-contraction (EC) coupling mechanism have been both proposed to contribute to the age-related decline of muscle performance. EC coupling in skeletal muscle fibers occurs at specialized intracellular junctions called calcium release units (CRUs), or triads. Recent findings demonstrated that in adult fast twitch skeletal fibers most inter-myofibrillar mitochondria are positioned at the I band and structurally coupled to CRUs. Here we have studied the morphology, frequency, and sarcomeric-localization of CRUs and mitochondria in extensor digitorum brevis (EDL) from ageing WT male mice using a combination of confocal and electron microscopy. Quantitative analysis revealed that: a) the number of CRUs/100mm² in aging mice (n=5, 25–35 months) decreases compared to adult mice (n=5, 3–12 months): 93±9 vs. 76±8, respectively; and b) also the number of mitochondria/100mm² decrease with age: 54±7 vs. 43±6 respectively.. In addition, both histological sections and immunofluorescence experiments suggest a partial misplacements of mitochondria from their normal location. All these changes taken together cause a large decrease in the number of functional CRUs-mitochondria couples in ageing: 39±5 vs. 26±5: a decrease of about 33%. The reduction of CRU/mitochondria couples may significantly contribute to the age-related decrease of specific force and endurance affecting skeletal muscle of elderly individuals.