Chemical hypoxia produces depletion of ATP, intracellular Ca2+ overload, and cell death. The role of Na+/Ca2+ exchanger (NCX), the major plasma membrane Ca2+ extruding system, has been explored in chemical hypoxia using BHK cells stably transfected with the three mammalian NCX isoforms: NCX1, NCX2, and NCX3. Here we report that the three isoforms show similar activity evaluated as [Ca2+]i increase evoked by Na+-free medium exposure in Fura-2-loaded single cells and NCX3 transfected cells are less vulnerable to chemical hypoxia compared to NCX1- and NCX2-transfected cells, suggesting that NCX3 could play a more relevant protective role during chemical hypoxia.