cADP-Ribose (cADPR) is a novel endogenous messenger that is believed to mobilize Ca(2+) from ryanodine-sensitive Ca(2+) stores. Despite intense research, the precise mechanism of action of cADPR remains uncertain, and experimental findings are contradictory. To elucidate the mechanism of cADPR action, we performed confocal Ca(2+) imaging in saponin-permeabilized rat ventricular myocytes. Exposure of the cells to cADPR resulted in a slow (>2 minutes) and steady increase in the frequency of Ca(2+) sparks. These effects on local release events were accompanied by a significant increase in sarcoplasmic reticulum (SR) Ca(2+) content. In comparison, sensitization of ryanodine receptors (RyRs) by caffeine, a true RyR agonist, caused a rapid (<1 second) and transient potentiation of Ca(2+) sparks followed by a decrease in SR Ca(2+) content. When the increase in the SR load was prevented by partial inhibition of the SR Ca(2+) with thapsigargin, cADPR failed to produce any increase in sparking activity. cADPR had no significant impact on activity of single cardiac RyRs incorporated into lipid bilayers. However, it caused a significant increase in the rate of Ca(2+) uptake by cardiac SR microsomes. Our results suggest that the primary target of cADPR is the SR Ca(2+) uptake mechanism. Potentiation of Ca(2+) release by cADPR is mediated by increased accumulation of Ca(2+) in the SR and subsequent luminal Ca(2+)-dependent activation of RyRs.