The excitation–contraction coupling cycle in cardiac muscle is initiated by an influx of Ca²⁺ through voltage-dependent Ca²⁺ channels. Ca²⁺ influx induces a release of Ca²⁺ from the sarcoplasmic reticulum and myocyte contraction. To maintain Ca²⁺ homeostasis, Ca²⁺ entry is balanced by efflux mediated by the sarcolemmal Na⁺-Ca²⁺ exchanger. In the absence of Na⁺-Ca²⁺ exchange, it would be expected that cardiac myocytes would overload with Ca²⁺. Using Cre/loxP technology, we generated mice with a cardiac-specific knockout of the Na⁺-Ca²⁺ exchanger, NCX1. The exchanger is completely ablated in 80% to 90% of the cardiomyocytes as determined by immunoblot, immunofluorescence, and exchange function. Surprisingly, the NCX1 knockout mice live to adulthood with only modestly reduced cardiac function as assessed by echocardiography. At 7.5 weeks of age, measures of contractility are decreased by 20% to 30%. We detect no adaptation of the myocardium to the absence of the Na⁺-Ca²⁺ exchanger as measured by both immunoblots and microarray analysis. Ca²⁺ transients of isolated myocytes from knockout mice display normal magnitudes and relaxation kinetics and normal responses to isoproterenol. Under voltage clamp conditions, the current through L-type Ca²⁺ channels is reduced by 50%, although the number of channels is unchanged. An abbreviated action potential may further reduce Ca²⁺ influx. Rather than upregulate other Ca²⁺ efflux mechanisms, the myocardium appears to functionally adapt to the absence of the Na⁺-Ca²⁺ exchanger by limiting Ca²⁺ influx. The magnitude of Ca²⁺ transients appears to be maintained by an increased gain of sarcoplasmic reticular Ca²⁺ release. The myocardium of the NCX1 knockout mice undergoes a remarkable adaptation to maintain near normal cardiac function.