Phosphorus magnetic resonance spectroscopy offers a unique opportunity to measure in vivo the free cytosolic magnesium [Mg2+] of different tissues. In particular, this technique has been employed in human brain and in skeletal muscle providing new hints on Mg2+ homeostasis and on its involvement in cellular bioenergetics. In skeletal muscle it has been shown that the changes of free Mg2+ concentration occurring during contraction and in post-exercise recovery are mainly due to the cytosolic pH influence. The possibility of assessing the free cytosolic [Mg2+] in the human brain offered the chance of studying the involvement of Mg2+ in different neurological pathologies, and particularly in those where defective mitochondrial energy production represents the primary causative factor in the pathogenesis. The results obtained, studying patients affected by different types of mitochondrial cytopathies, helped to clarify the functional relationship between energy metabolism and free [Mg2+], providing evidence that cytosolic [Mg2+] is regulated in brain cells to equilibrate any changes in rapidly available free energy. Moreover, it has also been shown that the measurement of brain Mg2+ can help in the differential diagnosis of neurodegenerative diseases sharing common clinical features, such as Multiple System Atrophy and Parkinson's disease.