—To elucidate microscopic mechanisms underlying the modulation of cardiac excitation-contraction (EC) coupling by β-adrenergic receptor (β-AR) stimulation, we examined local Ca²⁺ release function, ie, Ca²⁺ spikes at individual transverse tubule–sarcoplasmic reticulum (T-SR) junctions, using confocal microscopy and our recently developed technique for release flux measurement. β-AR stimulation by norepinephrine plus an α₁-adrenergic blocker, prazosin, increased the amplitude of SR Ca²⁺ release flux (J_SR), its running integral (∫J_SR), and L-type Ca²⁺ channel current (I_Ca), and it shifted their bell-shaped voltage dependence leftward by ≈10 mV, with the relative effects ranking I_Ca> J_SR>∫J_SR. Confocal imaging revealed that the bell-shaped voltage dependence of SR Ca²⁺ release is attributable to a graded recruitment of T-SR junctions as well as to changes in Ca²⁺ spike amplitudes. β-AR stimulation increased the fractional T-SR junctions that fired Ca²⁺ spikes and augmented Ca²⁺ spike amplitudes, without altering the SR Ca²⁺ load, suggesting that more release units were activated synchronously among and within T-SR junctions. Moreover, β-AR stimulation decreased the latency and temporal dispersion of Ca²⁺ spike occurrence at a given voltage, delivering most of the Ca²⁺ at the onset of depolarization rather than spreading it out throughout depolarization. Because the synchrony of Ca²⁺ spikes affects Ca²⁺ delivery per unit of time to contractile myofilaments, and because the myofilaments display a steep Ca²⁺ dependence, our data suggest that synchronization of SR Ca²⁺ release represents a heretofore unappreciated mechanism of β-AR modulation of cardiac inotropy.