Mitochondria contain four distinct compartments and a selectively permeable inner membrane. These compartments provide an important means of regulation of mitochondrial activity via various carrier and translocase activities. Nutritional substrates derived from glucose, fatty acids, and branched chain amino acids are utilized by mitochondria in a tightly controlled manner. This control is provided by the specific dehydrogenase enzymes and carnitine related reactions and is dependent on cellular needs for energy and availability of various substrates. Several possible in vivo controllers of mitochondrial respiration exist. In this review we have examined: the dependence of mitochondrial function on oxygen availability, the cellular phosphate potential, and the adenine nucleotide translocase activity; the role of adenosine as the regulator of coronary flow providing a link to mitochondrial metabolism; the importance of the creatine-phosphate shuttle and the existence of separate mitochondrial and myofibrillar creatine-kinase enzymes; and the physiologically important mechanisms of the regulation of mitochondrial metabolic activity in the heart.