Reactive oxygen species (ROS) and Ca(2+) signals are closely associated with the pathogenesis of cardiac hypertrophy. However, the cause and effect of the two signals in cardiac hypertrophy remain to be clarified. We extend our recent report by investigating a potential interaction between ROS and Ca(2+) signals utilizing in vitro and in vivo angiotensin II (ANG II)-induced cardiac hypertrophy models. ANG II-induced initial Ca(2+) transients mediated by inositol trisphosphate (IP(3)) triggered initial ROS production in adult rat cardiomyocytes. The ROS generated by activation of the NAD(P)H oxidase complex via Rac1 in concert with Ca(2+) activates ADP-ribosyl cyclase to generate cyclic ADP-ribose (cADPR). This messenger-mediated Ca(2+) signal further augments ROS production, since 2,2'-dihydroxyazobenzene, an ADP-ribosyl cyclase inhibitor, or 8-Br-cADPR, an antagonistic analog of cADPR, abolished further ROS production. Data from short hairpin RNA (shRNA)-mediated knockdown of Akt1 and p47(phox) demonstrated that Akt1 is the upstream key molecule responsible for the initiation of Ca(2+) signal that activates p47(phox) to generate ROS in cardiomyocytes. Nuclear translocation of nuclear factor of activated T-cell in cardiomyocytes was significantly suppressed by treatment with NAD(P)H oxidase inhibitors as well as by shRNA against Akt1 and p47(phox). Our results suggest that in cardiomyocytes Ca(2+) and ROS messengers generated by ANG II amplify the initial signals in a cooperative manner, thereby leading to cardiac hypertrophy.