1. To define the sub-cellular mechanisms of modulation of cardiac excitation-contraction (E-C) coupling by the beta-adrenergic pathway, we carried out confocal Ca(2+) imaging in conjunction with recordings of inward Ca(2+) current in fluo-3-loaded patch-clamped rat ventricular myocytes. 2. Isoproterenol (isoprenaline; ISO) increased the amplitude of the inward Ca(2+) current and the globally measured intracellular Ca(2+) transients. The gain of calcium-induced calcium release (CICR) was increased at all membrane potentials but especially at positive membrane potentials (> +30 mV). ISO dramatically broadened the bell-shaped voltage dependence of intracellular Ca(2+) transients by shifting the descending portion of the curve to very high membrane potentials. 3. The number of local release events (solitary sparks and conglomerates of overlapping sparks) induced by depolarizing steps to +30 mV was increased significantly by ISO. This potentiation of events was due to increased trigger calcium current (I(Ca)) as well the enhanced ability of I(Ca) to induce release. The amplitude and duration of solitary sparks were increased in the presence of ISO. In addition, ISO dramatically increased the proportion and the size ('mass') of clustered events. 4. Exclusion of Na(+) from the intra- and extracellular solutions prevented ISO from enhancing the ability of I(Ca) to trigger sparks. 5. We conclude that beta-adrenergic stimulation enhances the gain of the CICR cascade by increasing the fidelity of dihydropyridine receptor (DHPR)--ryanodine receptor (RyR) coupling and by promoting cross-activation of RyRs in neighbouring release sites. Reverse Na(+)--Ca(2+) exchange (NCX) appears to play a role in the beta-adrenergic enhancement of CICR by effectively contributing to the Ca(2+) trigger.