Bi-directional calcium (Ca(2+)) signaling between mitochondria and intracellular stores (endoplasmic/sarcoplasmic reticulum) underlies important cellular functions, including oxidative ATP production. In striated muscle, this coupling is achieved by mitochondria being located adjacent to Ca(2+) stores (sarcoplasmic reticulum [SR]) and in proximity of release sites (Ca(2+) release units [CRUs]). However, limited information is available with regard to the mechanisms of mitochondrial-SR coupling. Using electron microscopy and electron tomography, we identified small bridges, or tethers, that link the outer mitochondrial membrane to the intracellular Ca(2+) stores of muscle. This association is sufficiently strong that treatment with hypotonic solution results in stretching of the SR membrane in correspondence of tethers. We also show that the association of mitochondria to the SR is 1) developmentally regulated, 2) involves a progressive shift from a longitudinal clustering at birth to a specific CRU-coupled transversal orientation in adult, and 3) results in a change in the mitochondrial polarization state, as shown by confocal imaging after JC1 staining. Our results suggest that tethers 1) establish and maintain SR-mitochondrial association during postnatal maturation and in adult muscle and 2) likely provide a structural framework for bi-directional signaling between the two organelles in striated muscle.