Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca-releasing second messenger known to date. Here, we report a new role for NAADP in arrhythmogenic Ca release in cardiac myocytes evoked by β-adrenergic stimulation. Infusion of NAADP into intact cardiac myocytes induced global Ca signals sensitive to inhibitors of both acidic Ca stores and ryanodine receptors and to NAADP antagonist BZ194. Furthermore, in electrically paced cardiac myocytes BZ194 blocked spontaneous diastolic Ca transients caused by high concentrations of the β-adrenergic agonist isoproterenol. Ca transients were recorded both as increases of the free cytosolic Ca concentration and as decreases of the sarcoplasmic luminal Ca concentration. Importantly, NAADP antagonist BZ194 largely ameliorated isoproterenol-induced arrhythmias in awake mice. We provide strong evidence that NAADP-mediated modulation of couplon activity plays a role for triggering spontaneous diastolic Ca transients in isolated cardiac myocytes and arrhythmias in the intact animal. Thus, NAADP signaling appears an attractive novel target for antiarrhythmic therapy.