New discoveries in the last decade significantly altered our view on mitochondria. They are no longer viewed as energy-making slaves but rather individual cells-within-the-cell. In particular, it has been suggested that many important cellular mechanisms involving specific enzymes and ion channels, such as nitric oxide synthase (NOS), ATP-dependent K+ (KATP) channels, and poly-(APD-ribose) polymerase (PARP), have a distinct, mitochondrial variant. Unfortunately, exploring these parallel systems in mitochondria have technical limitations and inappropriate methods often led to inconsistent results. For example, the intriguing possibility that mitochondria are significant sources of nitric oxide (NO) via a unique mitochondrial NOS variant has attracted intense interest among research groups because of the potential for NO to affect functioning of the electron transport chain. Nonetheless, conclusive evidence concerning the existence of mitochondrial NO synthesis is yet to be presented. This review summarizes the experimental evidence gathered over the last decade in this field and highlights new areas of research that reveal surprising dimensions of NO production and metabolism by mitochondria.