1. We examined [Ca2+]i and L-type Ca2+ channel current (ICa) in single cardiac myocytes to determine how the intracellular protein phospholamban (PLB) influences excitation-contraction (E-C) coupling in heart. Wild type (WT) and PLB-deficient (KO) mice were used. Cells were patch clamped in whole-cell mode while [Ca2+]i was imaged simultaneously using the Ca2+ indicator fluo-3 and a confocal microscope. 2. Although ICa was similar in magnitude, the decay of ICa was faster in KO than in WT cells and the [Ca2+]i transient was larger and decayed faster. Furthermore, the E-C coupling 'gain' (measured as delta[Ca2+]i/ICa) was larger in KO cells than in WT cells. 3. Spontaneous Ca2+ sparks were three times more frequent and larger in KO cells than in WT myocytes but, surprisingly, the time constants of decay were similar. 4. SR Ca2+ content was significantly greater in KO than in WT cells. When the SR Ca2+ content in KO cells was reduced to that in WT cells, Ca2+ sparks in these 'modified' (KO') cells decayed faster. E-C coupling gain, [Ca2+]i transient amplitude and the kinetics of decay of ICa were similar in KO' and WT cells. 5. We conclude that SR Ca2+ content influences (1) ICa, (2) the amplitude and kinetics of Ca2+ sparks and [Ca2+]i transients, (3) the sensitivity of the RyRs to triggering by [Ca2+]i, (4) the amount of Ca2+ released, (5) the magnitude of the E-C coupling 'gain' function, and (6) the rate of Ca2+ re-uptake by the SR Ca(2+)-ATPase. In KO cells, the larger [Ca2+]i transients and Ca2+ sparks speed up ICa inactivation. Finally, we conclude that PLB plays an important regulatory role in E-C coupling by modulating SR Ca(2+)-ATPase activity, which establishes the SR Ca2+ content and consequently influences the characteristics of local and global Ca2+ signalling.