Na+-Ca2+exchange has been shown to contribute to reperfusion- and reoxygenation-induced cellular Ca2+loading and damage in the heart. Despite the fact that both [Na+]iand [Ca2+]ihave been documented to rise during ischemia and hypoxia, it remains unclear whether the rise in [Ca2+]ioccurring during hypoxia is linked to the rise in [Na+]ivia Na+-Ca2+exchange before reoxygenation and how this relates to cellular injury. Single electrically stimulated (0.2 Hz) adult rat cardiac myocytes loaded with Na+-sensitive benzofuran isophthalate (SBFI), the new fluorescent probe, were exposed to glucose-free hypoxia (Po2<0.02 mm Hg), and SBFI fluorescence was monitored to index changes in [Na+]i. Parallel experiments were performed with indo-1-loaded cells to index [Ca2+]i. The SBFI fluorescence ratio (excitation, 350/380 nm) rose significantly during hypoxia after the onset of ATP-depletion contracture, consistent with a rise in [Na+]i. At reoxygenation, the ratio fell rapidly toward baseline levels. The indo-1 fluorescence ratio (emission, 410/490 nm) also rose only after the onset of rigor contracture and then often showed a secondary rise early after reoxygenation at a time when [Na+]ifell. The increase in both [Na+]iand [Ca2+]i, seen during hypoxia, could be markedly reduced by performing experiments in Na+-free buffer. These experiments suggested that hypoxic Ca2+loading is linked to a rise in Na+ivia Na+-Ca2+exchange. To show that Na+-Ca2+exchange activity was not fully inhibited by profound intracellular ATP depletion, cells were exposed to cyanide, and then buffer Na+was abruptly removed after contracture occurred. The sudden removal of buffer Na+would be expected to stimulate cell Ca2+entry via Na+-Ca2+exchange. A large rapid rise in the indo-1 fluorescence ratio ensued, which was consistent with abrupt cell Ca2+loading via the exchanger. The effect of reducing hypoxic buffer [Na+] on cell morphology after reoxygenation was examined. Ninety-five percent of cells studied in a normal Na+-containing buffer (144 mM NaCl,n=38) and reoxygenated 30 minutes after the onset of hypoxic rigor underwent hypercontracture. Only 12% of cells studied in Na+-free buffer (144 mM choline chloride,n=17) hypercontracted at reoxygenation (p<0.05). Myocytes were also exposed to hypoxia in the presence of R 56865, a compound that blocks noninactivating components of the Na+current. R 56865 blunted the rise in [Na+]itypically seen after the onset of rigor, suggesting that Na+entry may occur, in part, through voltage-gated Na+channels. These experiments provide evidence that [Na+]irises during hypoxia and leads to cellular Ca2+loading and cell destruction via Na+-Ca2+exchange. Prevention of the rise in [Na+]iduring hypoxia reduces cellular injury in this model. Further studies are required to fully elucidate the mechanisms underlying the rise in [Na+]ithat occurs during hypoxia and ischemia.