Studies from this laboratory and others have established the important role of intracellular Ca 2+ in cardiac myocyte injury resulting from exposure to hypoxia. Cellular Ca 2+ loading is thought to result from Na+ loading occurring during hypoxia and subsequent exchange for Ca 2+ via Na+ Ca 2+ exchange at reoxygenation. We monitored [Na+] i and [Ca 2+] i in single rat cardiac myocytes during exposure to glucose-free hypoxia and reoxygenation using the ion sensitive fluorescent probes SBFI (Na+ sensitive) and indo-1 (Ca 2+ sensitive). Cells exposed to hypoxia showed a progressive marked rise in both [Na+] i and [Ca 2+] i following the onset of profound energy depletion (rigor contracture). At reoxygenation [Ca 2+] i often rose further as [Na+] i abruptly fell. By completely replacing buffer Na+ with choline we nearly prevented the rise in [Na+] i seen during hypoxia and attenuated the rise in [Ca 2+] i seen during the same period. Cell survival was significantly enhanced in this low Na+ protocol. These studies demonstrate for the first time that intracellular Ca 2+ loading during hypoxia occurs via Na+-Ca 2+ exchange and that prevention of Na+ loading enhances post-hypoxic cell recovery.