Inhibition by cardiac glycosides of Na+, K+-ATPase reduces sodium efflux from myocytes and may lead to Na+ and Ca2+ overload and detrimental effects on mechanical function, energy metabolism, and electrical activity. We hypothesized that inhibition of sodium persistent inward current (late INa) would reduce ouabain's effect to cause cellular Na+ loading and its detrimental metabolic (decrease of ATP) and functional (arrhythmias, contracture) effects. Therefore, we determined effects of ouabain on concentrations of intracellular sodium (Na+i) and high-energy phosphates using 23Na and 31P NMR, the amplitude of late INa using the whole-cell patch-clamp technique, and contractility and electrical activity of guinea pig isolated hearts, papillary muscles, and ventricular myocytes in the absence and presence of inhibitors of late INa. Ouabain (1–1.3 μM) increased Na+i and late INa of guinea pig isolated hearts and myocytes by 3.7- and 4.2-fold, respectively. The late INa inhibitors ranolazine and tetrodotoxin significantly reduced ouabain-stimulated increases in Na+i and late INa. Reductions of ATP and phosphocreatine contents and increased diastolic tension in ouabain-treated hearts were also markedly attenuated by ranolazine. Furthermore, the ouabain-induced increase of late INa was also attenuated by the Ca2+-calmodulin-dependent kinase I inhibitors KN-93 [N-[2-[[[3-(4-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulphonamide] and autocamide-2 related inhibitory peptide, but not by KN-92 [2-[N-(4′-methoxybenzenesulfonyl)]amino-N-(4′-chlorophenyl)-2-propenyl-N-methylbenzylamine phosphate]. We conclude that ouabain-induced Na+ and Ca2+ overload is ameliorated by the inhibition of late INa.