Although Na⁺-H⁺ exchanger (NHE) is considered to be involved in the regulation of intracellular Ca²⁺ through Na⁺-Ca²⁺ exchanger, the exact mechanisms of its participation in Ca²⁺-handling by cardiomyocytes are not fully understood. To gain some information in this regard, isolated rat cardiomyocytes were exposed to a NHE inhibitor, 5-(N-Methyl-N-isobutyl) amiloride (MIA), upon treatment with or without different agents, which are known to modify Ca²⁺-movements in cardiomyocytes. [Ca²⁺]_i in cardiomyocytes was measured spectrofluorometrically with fura-2 AM in the absence or presence of KCl, a depolarizing agent. MIA was found to increase the basal [Ca²⁺]_i and augment the KCl-induced increase in [Ca²⁺]_i in a concentration dependent manner. The MIA-induced increase in basal [Ca²⁺]_i was unaffected by extracellular Ca²⁺, antagonists of sarcolemmal (SL) L-type Ca²⁺ channel as well as inhibitors of SL Na⁺-Ca²⁺ exchanger, SL Ca²⁺-pump ATPase and mitochondrial Ca²⁺-uptake. However, the MIA-induced increase in basal [Ca²⁺]_i was attenuated by inhibitors of SL Na⁺-K⁺ ATPase and sarcoplasmic reticulum (SR) Ca²⁺-transport. On the other hand, the MIA-mediated augmentation of the KCl response was dependent on extracellular concentration of Ca²⁺ and attenuated by agents, which inhibit SL L-type Ca²⁺ channels, SL Na⁺-Ca²⁺ exchanger, SL Na⁺-K⁺ ATPase as well as SR Ca²⁺-release channels and SR Ca²⁺-pump. However, the effect of MIA on the KCl-induced increase in [Ca²⁺]_i remained unaffected by treatment with inhibitors of SL Ca²⁺-pump ATPase and mitochondrial Ca²⁺-uptake. Both MIA and a decrease in extracellular pH lowered the intracellular pH and increased the basal [Ca²⁺]_i whereas the decrease in extracellular pH, unlike MIA, depressed the KCl-induced increase in [Ca²⁺]_i in cardiomyocytes. These results suggest that NHE may be involved in the regulation of [Ca²⁺]_i and the MIA-induced increases in basal [Ca²⁺]_i as well as augmentation of KCl-induced increase in [Ca²⁺]_i in cardiomyocytes are regulated differentially.