Although low Na+is known to increase the intracellular Ca2+concentration ([Ca2+]i) in cardiac muscle, the exact mechanisms of low Na+-induced increases in [Ca2+]iare not completely defined. To gain information in this regard, we examined the effects of low Na+(35 mM) on freshly isolated cardiomyocytes from rat heart in the absence and presence of different interventions. The [Ca2+]iin cardiomyocytes was measured fluorometrically with Fura-2 AM. Following a 10 min incubation, the low Na+-induced increase in [Ca2+]iwas only observed in cardiomyocytes depolarized with 30 mM KCl, but not in quiescent cardiomyocytes. In contrast, low Na+did not alter the ATP-induced increase in [Ca2+]iin the cardiomyocytes. This increase in [Ca2+]idue to low Na+and elevated KCl was dependent on the extracellular concentration of Ca2+(0.25–2.0 mM). The L-type Ca2+-channel blockers, verapamil and diltiazem, at low concentrations (1 μM) depressed the low Na+, KCl-induced increase in [Ca2+]iwithout significantly affecting the response to low Na+alone. The low Na+, high KCl-induced increase in [Ca2+]iwas attenuated by treatments of cardiomyocytes with high concentrations of both verapamil (5 and 10 μM), and diltiazem (5 and 10 μM) as well as with amiloride (5–20 μM), nickel (1.25–5.0 mM), cyclopiazonic acid (25 and 50 μM) and thapsigargin (10 and 20 μM). On the other hand, this response was augmented by ouabain (1 and 2 mM) and unaltered by 5-(N-methyl-N-isobutyl) amiloride (5 and 10 μM). These data suggest that in addition to the sarcolemmal Na+−Ca2+exchanger, both sarcolemmal Na+−K+ATPase, as well as the sarcoplasmic reticulum Ca2+-pump play prominent roles in the low Na+-induced increase in [Ca2+]i. (Mol Cell Biochem 263:151–162, 2004)