The spatial relationship between mitochondria and the membrane systems, more specifically the calcium release units (CRUs) of skeletal muscle, is of profound functional significance. CRUs are the sites at which Ca(2+) is released from the sarcoplasmic reticulum during muscle activation. Close mitochondrion-CRU proximity allows the organelles to take up Ca(2+) and thus stimulate aerobic metabolism. Skeletal muscles of most mammals display an extensive, developmentally regulated, close mitochondrion-CRU association, fostered by tethering links between the organelles. A comparative look at the vertebrate subphylum however shows that this specific association is only present in the higher vertebrates (mammals). Muscles in all other vertebrates, even if capable of fast activity, rely on a less precise and more limited mitochondrion-CRU proximity, despite some tethering connections. This is most evident in fish muscles. Clustering of free subsarcolemmal mitochondria in proximity of capillaries is also more frequently achieved in mammalian than in other vertebrates.