Mitochondrial Na+/Ca2+ exchange (NCXmito) is critical for neuronal Ca2+ homeostasis and prevention of cell death from excessive mitochondrial Ca2+ (m[Ca2+]) accumulation. The mitochondrial kinase PINK1 appears to regulate the mCa2+ efflux from dopaminergic (DAergic) neurons, possibly by directly regulating NCXmito. However, the precise molecular identity of NCXmito is unknown and has been the subject of great controversy. Here we propose that the previously characterised plasmalemmal NCX isoforms (NCX2, NCX3) contribute to mitochondrial Na +/Ca2+ exchange in human DAergic neurons and may act downstream of PINK1 in the prevention of neurodegeneration by m[Ca2+] accumulation. Firstly, we definitively show the existence of a mitochondrial pool of endogenous plasmalemmal NCX isoforms in human DAergic neurons and cell lines using immunolocalisation and fluorescence-assisted organelle sorting (FAOS). Secondly, we demonstrate reduced mitochondrial Ca2+ efflux occurs following inhibition of NCX2 or NCX3 (but not NCX1) using siRNA or antibody blocking. This study has potentially revealed a new molecular target in Parkinson's disease pathology which ultimately may open up new avenues for future therapeutic intervention. © Springer Science+Business Media New York 2013.