Parkinson's disease (PD), the second most common neurodegenerative disorder, is characterized by dopaminergic neuronal loss that initiates in the substantia nigra pars compacta and by the formation of intracellular inclusions mainly constituted by aberrant α-synuclein (α-syn) deposits known as Lewy bodies. Most cases of PD are sporadic, but about 10% are familial, among them those caused by mutations in SNCA gene have an autosomal dominant transmission. SNCA encodes α-syn, a small 140-amino acids protein that, under physiological conditions, is mainly localized at the presynaptic terminals. It is prevalently cytosolic, but its presence has been reported in the nucleus, in the mitochondria and, more recently, in the mitochondria-associated ER membranes (MAMs). Whether different cellular localizations may reflect specific α-syn activities is presently unclear and its action at mitochondrial level is still a matter of debate. Mounting evidence supports a role for α-syn in several mitochondria-derived activities, among which maintenance of mitochondrial morphology and modulation of complex I and ATP synthase activity. α-syn has been proposed to localize at the outer membrane (OMM), in the intermembrane space (IMS), at the inner membrane (IMM) and in the mitochondrial matrix, but a clear and comparative analysis of the sub-mitochondrial localization of WT and mutant α-syn is missing. Furthermore, the reasons for this spread sub-mitochondrial localization under physiological and pathological circumstances remain elusive. In this context, we decided to selectively monitor the sub-mitochondrial distribution of the WT and PD-related α-syn mutants A53T and A30P by taking advantage from a bimolecular fluorescence complementation (BiFC) approach. We also investigated whether cell stress could trigger α-syn translocation within the different mitochondrial sub-compartments and whether PD-related mutations could impinge on it. Interestingly, the artificial targeting of α-syn WT (but not of the mutants) to the mitochondrial matrix impacts on ATP production, suggesting a potential role within this compartment.