Phospholemman (PLM) regulates contractility and Ca(2+) homeostasis in cardiac myocytes. We characterized excitation-contraction coupling in myocytes isolated from PLM-deficient mice backbred 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 with wild-type myocytes, Western blots indicated total absence of PLM but no changes in Na(+)/Ca(2+) exchanger, sarcoplasmic reticulum (SR) Ca(2+)-ATPase, alpha(1)-subunit of Na(+)-K(+)-ATPase, and calsequestrin levels in PLM-null myocytes. At 5 mM extracellular Ca(2+) concentration ([Ca(2+)](o)), contraction and cytosolic [Ca(2+)] ([Ca(2+)](i)) transient amplitudes and SR Ca(2+) contents in PLM-null myocytes were significantly (P < 0.0004) higher than wild-type myocytes, whereas the converse was true at 0.6 mM [Ca(2+)](o). This pattern of contractile and [Ca(2+)](i) transient abnormalities in PLM-null myocytes mimics that observed in adult rat myocytes overexpressing the cardiac Na(+)/Ca(2+) exchanger. Indeed, we have previously reported that Na(+)/Ca(2+) exchange currents were higher in PLM-null myocytes. Activation of protein kinase A resulted in increased inotropy 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 Ca(2+) 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 Ca(2+) fluxes, likely by modulating Na(+)/Ca(2+) exchange activity.