Mitochondrial biochemistry is complex, expanding from oxidative phosphorylation, lipid catabolism and haem biosynthesis, to apoptosis, calcium homeostasis, and production of reactive oxygen and nitrogen species, including nitric oxide (NO). This molecule is produced by a mitochondrial nitric-oxide synthase (mtNOS). The rates of consumption and production determine the steady-state concentration of NO at subcellular levels, leading to the regulation of several mitochondrial events. Temporospatial processes tightly regulate the production of NO in mitochondria to maximize target effects and minimize deleterious reactions. Temporal regulatory mechanisms of mtNOS include activation by calcium and transcriptional/translational regulation. Calcium-activated mtNOS inhibits mitochondrial respiration. This regulation antagonizes the effects of calcium on matrix calcium-dependent dehydrogenases, preventing the formation of anoxic foci. Temporal regulation of NO production by intracellular calcium signalling requires the understanding of the heterogeneous intracellular calcium response and calcium distribution. NO production in mitochondria is spatially regulated by subcellular localization of mtNOS (e.g. acylation and protein-protein interactions), in addition to transcriptional regulation. Given the short half-life of NO in biological systems, organelle localization of mtNOS is crucial for NO to function as a signal molecule. These temporospatial processes are biologically important to allow NO to act as an effective signal molecule to regulate mitochondrial events.