Abstract—Spontaneous sarcoplasmic reticulum (SR) Ca2+release causes delayed afterdepolarizations (DADs) via Ca2+-induced transient inward currents (Iti). However, no quantitative data exists regarding (1) Ca2+dependence of DADs, (2) Ca2+required to depolarize the cell to threshold and trigger an action potential (AP), or (3) relative contributions of Ca2+-activated currents to DADs. To address these points, we evoked SR Ca2+release by rapid application of caffeine in indo 1-AM–loaded rabbit ventricular myocytes and measured caffeine-induced DADs (cDADs) with whole-cell current clamp. The SR Ca2+load of the myocyte was varied by different AP frequencies. The cDAD amplitude doubled for every 88±8 nmol/L of &Dgr;[Ca2+]i(simple exponential), and the &Dgr;[Ca2+]ithreshold of 424±58 nmol/L was sufficient to trigger an AP. Blocking Na+-Ca2+exchange current (INa/Ca) by removal of [Na]oand [Ca2+]o(or with 5 mmol/L Ni2+) reduced cDADs by >90%, for the same &Dgr;[Ca2+]i. In contrast, blockade of Ca2+-activated Cl–current (ICl(Ca)) with 50 &mgr;mol/L niflumate did not significantly alter cDADs. We conclude that DADs are almost entirely due toINa/Ca, notICl(Ca)or Ca2+-activated nonselective cation current. To trigger an AP requires 30 to 40 &mgr;mol/L cytosolic Ca2+or a [Ca2+]itransient of 424 nmol/L. Current injection, simulatingItis with different time courses, revealed that fasterItis require less charge for AP triggering. Given that spontaneous SR Ca2+release occurs in waves, which are slower than cDADs or fastItis, the true &Dgr;[Ca2+]ithreshold for AP activation may be ≈3-fold higher in normal myocytes. This provides a safety margin against arrhythmia in normal ventricular myocytes.