Although sarcolemmal (SL) Na⁺-Ca²⁺ exchanger is known to regulate the intracellular Ca²⁺ concentration ([Ca²⁺]_i), its involvement in catecholamine-induced increase in [Ca²⁺]_i is not fully understood. To gain some information in this regard, isolated rat cardiomyocytes were treated with different agents, which are known to modify Ca²⁺-movements, in the absence or presence of a B-adrenoceptor agonist, isoproterenol, and [Ca²⁺]_i in cardiomyocytes was determined spectrofluorometrically with Fura-2 AM. Treatment with isoproterenol did not alter [Ca²⁺]_i in quiescent cardiomyocytes, whereas the ATP, a purinergic receptor agonist, induced increase in [Ca²⁺]_i was significantly potentiated by isoproterenol. Unlike ryanodine and cyclopiazonic acid, which affect the sarcoplasmic reticulum function, SL L-type Ca²⁺ channel blockers, verapamil and diltiazem, as well as a SL Ca²⁺-pump inhibitor, vanadate, caused a significant depression in the isoproterenol-induced increase in [Ca²⁺]_i. The SL Na⁺-Ca²⁺ exchange blockers, amiloride, Ni²⁺ and KB-R7943, also attenuated the isoproterenol mediated increase in [Ca²⁺]_i.Combination of KB-R7943 and verapamil showed additive inhibitory effects on the isoproterenol-induced increase in [Ca²⁺]_i.The isoproterenol-induced increase in [Ca²⁺]_i in KCl- depolarized cardiomyocytes, unlike that in the presence of ATP, was augmented by low Na⁺; this augmentation was significantly depressed by treatment with KB-R7943. The positive inotropic action of isoproterenol in isolated hearts was also reduced by KB-R7943. These data suggest that in addition to SL L-type Ca²⁺ channels, SL Na⁺-Ca²⁺ exchanger seems to play an important role in catecholamine-induced increase in [Ca²⁺]_i in cardiomyocytes.