The novel inhibitor of the reverse mode of the Na+/Ca2+ exchanger (NCE) KB-R7943 (KB) was tested in isolated rat cardiomyocytes exposed to 80 min of simulated ischemia [substrate-free anoxia, extracellular pH (pHo) of 6.4] and 15 min of reoxygenation (pHo 7.4). At pHo 6.4, 20 micromol/l KB was required for complete inhibition of the reverse mode of NCE. Treatment with 20 micromol/l KB only during anoxia did not influence the onset of rigor contracture and intracellular pH (pHi) (monitored with 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein) but significantly reduced the cytosolic accumulation of Ca2+ (monitored with fura 2) and Na+ (monitored with sodium-binding benzofuran isophthalate). During reoxygenation, cardiomyocytes developed hypercontracture. This was significantly reduced by anoxic KB treatment. To investigate this protection against reoxygenation-induced injury in the whole heart, we exposed Langendorff-perfused rat hearts to 110 min of anoxia (pHo 6.4) and 50 min of reoxygenation (pHo 7.4). Application of 20 micromol/l KB during anoxia significantly reduced the reoxygenation-induced enzyme release. We conclude that KB offers significant protection of cardiomyocytes against Ca2+ and Na+ overload during anoxia and hypercontracture or enzyme release on reoxygenation.