Although inhibition of the sarcolemmal (SL) Na⁺-K⁺ ATPase ATPase is known to cause an increase in the intracellular concentration of Ca²⁺ ([Ca²⁺]_i) by stimulating the SL [Na⁺]_i -Ca²⁺ exchanger (NCX), the involvement of other SL sites in inducing this increase in [Ca²⁺]_i is not fully understood. Isolated rat cardiomyocytes were treated with or without different agents, which modify Ca²⁺-movements by affecting various SL sites before exposure to ouabain. Ouabain was observed to increase the basal levels of both [Ca²⁺]_i and intracellular Na⁺ concentration ([Na⁺]_i ) as well as augmented the KCl-induced increases in both [Ca²⁺]_i and [Na⁺]_i in a concentration dependent manner. The ouabain-induced changes in [Na⁺]_i and [Ca²⁺]_i were attenuated by treatment with inhibitors of SL Na⁺-K⁺ ATPase exchanger (NHE) and SL Na⁺-channels. Both ouabain-induced increase in basal [Ca²⁺]_i and augmentation of KCl response were markedly decreased when cardiomyocytes were exposed to 0 to 10 mM Na⁺. Inhibitors of SL NCX depressed whereas decreasing extracellular Na⁺ from 105 to 35 mM augmented the ouabain-induced increase in basal [Ca²⁺]_i and KCl response. Not only the increase in [Ca²⁺]_i by ouabain was dependent on the extracellular Ca²⁺ concentration but was also attenuated by inhibitors of SL L-type Ca²⁺ channels and store-operated Ca²⁺ channels (SOC). Unlike the SL L-type Ca²⁺-channel blocker, the blockers of SL Na⁺ channel and SL SOC, when used in combination with SL NCX inhibitor, showed additive effects in reducing the ouabain-induced increase in basal [Ca²⁺]_i. These results support the view that in addition to SL NCX, SL L-type Ca²⁺ channels and SL SOC may be involved in raising the [Ca²⁺]_i upon inhibiting the SL Na⁺-K⁺ ATPase ATPase by ouabain. Furthermore, both SL NHE and Na⁺ channels play a critical role in the ouabain-induced Ca²⁺ increase in cardiomyocytes.