ADP-ribosyl cyclase (ADPR-cyclase) produces a Ca2+-mobilizing second messenger cyclic ADP-ribose (cADPR) from beta-NAD(+). In this study, we examined the molecular basis of which beta-adrenergic receptor (beta AR) stimulation induces cADPR formation and characterized cardiac ADPR-cyclase. The results revealed that isoproterenol-mediated increase of [Ca2+](i) in rat cardiomyocytes was blocked by pretreatment with a cADPR antagonistic derivative 8-Bi-cADPR, a PKA inhibitor H89 or high concentration of ryanodine. Moreover, incubation of ventricular lysates with isoproterenol, forskolin or cAMP resulted in activation of ADPR-cyclase that was inhibited by pretreatment with H89. Supporting the observations, the cADPR antagonist and H89 blocked 8-CPT-cAMP, a cell-permeant cAMP analog-induced increase in [Ca2+](i) but not cGMP-mediated increase. Characterization of partially purified cardiac ADPR-cyclase showed a molecular mass of approximately 42 kDa and no cross-activity with CD38 antibodies, and the enzyme activity was inhibited by Zn2+ but not dithiothreitol. Microinjection of the enzyme into rat cardiomyocytes increased the level of [Ca2+](i) in a concentration-dependent manner. The enzyme-mediated increase of [Ca2+](i) was blocked by the cADPR antagonist. These findings suggest that beta AR-mediated regulation of [Ca2+](i) in rat cardiomyocytes is primed by activation of cardiac ADPR-cyclase via cAMP/PKA signaling and that cardiac ADPR-cyclase differs from CD38 in biochemical and immunological properties. (c) 2005 Elsevier Inc. All rights reserved.