Phospholemman (PLM), a 72-amino acid sarcolemmal protein, has been shown to regulate contractility and Ca²⁺ homeostasis in cardiac myocytes. We characterized excitation-contraction coupling in myocytes isolated from PLM-deficient mice back-bred to a pure congenic C57BL/6 background. Cell length, cell width and whole cell capacitance were not different between wild-type and PLM-null myocytes. Compared to wild-type myocytes, Western blots indicated total absence of PLM but no changes in Na⁺/Ca²⁺ exchanger, sarcoplasmic reticulum (SR) Ca²⁺-ATPase, 1-subunit of Na⁺-K⁺-ATPase and calsequestrin levels in PLM-null myocytes. At 5mM extracellular [Ca²⁺] concentration ([Ca²⁺]_o), contraction and cytosolic [Ca²⁺] ([Ca²⁺]_i) transient amplitudes and SR [Ca²⁺] contents in PLM-null myocytes were significantly (p<0.0004) higher than wild-type myocytes. At 0.6 mM [Ca²⁺]_o, however, contraction and [Ca²⁺]_i transient amplitudes and SR [Ca²⁺] contents were significantly lower in PLM-null than wild-type myocytes. At 1.8 mM [Ca²⁺]_o, the differences in contraction and [Ca²⁺]_i transient amplitudes were no longer apparent. This pattern of contractile and [Ca²⁺]_i transient abnormalities in PLM-null myocytes mimics that observed in adult rat myocytes overexpressing the cardiac Na⁺/Ca²⁺ exchanger. Indeed, we have previosuly reported that Na⁺/Ca²⁺ exchange currents were higher in PLM-null myocytes. Activation of protein kinase A resulted in increased inotrophy such that there were no longer any contractility differences between the stimulated wild-type and PLM-null myocytes. Protein kinase C stimulation resulted in decreased contractility in both wild-type and PLM-null myocytes. Resting membrane potential and action potential amplitudes were similar, but action potential duration was much prolonged (p<0.04) in PLM-null myocytes. Whole cell Ca²⁺ current densities were similar between wild-type and PLM-null myocytes, as were the fast and slow inactivation time constants. We conclude that a major function of PLM is regulation of cardiac contractility and Ca²⁺ fluxes, likely by modulating Na⁺/Ca²⁺ exchange activity.