Bi-directional Ca²⁺ signaling between mitochondria and intracellular stores (sarcoplasmic reticulum, SR) is crucial for fundamental functions of skeletal fibers, including oxidative ATP production. However, the Ca²⁺ concentration required for mitochondrial uptake (Kd=~30 mM) is much higher than that typically achieved during cytosolic Ca²⁺ transients (~1 mM). This apparent discrepancy is reconciled by the concept of Ca²⁺ microdomains: strategic positioning of mitochondria in proximity of Ca²⁺ sources (Ca²⁺ release units, CRUs) is likely required to allow cross-talk between the two organelles. We characterized the mechanisms responsible for orchestrating and maintaining mitochondria/CRU proximity during postnatal development: association of mitochondria with CRUs is a highly coordinated process that involves a progressive migration of mitochondria at the I band, next to CRUs. Mitochondrial-CRU coupling is possible thanks to small strands, or tethers, bridging the outer mitochondrial membrane to the SR. An overview of skeletal muscle over the entire vertebrate subphylum reveals that this restricted positioning of mitochondria is an exclusive property of mammalian skeletal muscle. Interestingly, mitochondria-CRU association is affected by several conditions in which Ca²⁺ handling is de-regulated, i.e. animal models carrying mutations of proteins involved in excitation-contraction coupling, or in physio-pathological conditions such as ageing and denervation.