The specific role played by NCX1, NCX2, and NCX3, the three isoforms of the Na+/Ca2+ exchanger (NCX), has been explored during hypoxic conditions in BHK cells stably transfected with each of these isoforms. Six major findings emerged from the present study: (1) all the three isoforms were highly expressed on the plasma membranes of BHK cells; (2) under physiological conditions, the three NCX isoforms showed similar functional activity; (3) hypoxia plus reoxygenation induced a lower increase of [Ca2+]i in BHK-NCX3-transfected cells than in BHK-NCX1- and BHK-NCX2-transfected cells; (4) NCX3-transfected cells were more resistant to chemical hypoxia plus reoxygenation than NCX1- and NCX2-transfected cells. Interestingly, such augmented resistance was eliminated by CBDMD (10 microM), an inhibitor of NCX and by the specific silencing of the NCX3 isoform; (5) chemical hypoxia plus reoxygenation produced a loss of mitochondrial membrane potential in NCX1- and NCX2-transfected cells, but not in NCX3-transfected cells; (6) the forward mode of operation in NCX3-transfected cells was not affected by ATP depletion, as it occurred in NCX1- and NCX2-transfected cells. Altogether, these results indicate that the brain specifically expressed NCX3 isoform more significantly contributes to the maintenance of [Ca2+]i homeostasis during experimental conditions mimicking ischemia, thereby preventing mitochondrial delta psi collapses and cell death.