The recent discovery of mitochondrial nitric-oxide synthase (mtNOS) has provided novel information on mitochondrial biology. mtNOS, localized at the inner membrane, functions as one of the important regulatory factors that affect cellular respiration in mitochondria. The distribution of mtNOS in diverse organs of many species suggests its biological importance. Changes in mtNOS expression and activity in many pathophysiological situations may imply its significant involvement in various NO-related biological phenomena. The functional coordination of mtNOS with other NOSs present in the same or adjacent tissues is unknown as it is most of the regulatory mechanisms of mtNOS expression. Thus, future studies will be required to validate the physiological significance of mtNOS, and to elucidate its regulatory mechanisms on cellular energy metabolism in mitochondria.