—Cardiac beating arises from the spontaneous rhythmic excitation of sinoatrial (SA) node cells. Here we report that SA node pacemaker activity is critically dependent on Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). In freshly dissociated rabbit single SA node cells, inhibition of CaMKII by a specific peptide inhibitor, autocamtide-2 inhibitory peptide (AIP, 10 μmol/L), or by KN-93 (0.1 to 3.0 μmol/L), but not its inactive analog, KN-92, depressed the rate and amplitude of spontaneous action potentials (APs) in a dose-dependent manner. Strikingly, 10 μmol/L AIP and 3 μmol/L KN-93 completely arrested SA node cells, which indicates that basal CaMKII activation is obligatory to the genesis of pacemaker AP. To understand the ionic mechanisms of the CaMKII effects, we measured L-type Ca²⁺ current (I_{Ca, L}), which contributes both to AP upstroke and to pacemaker depolarization. KN-93 (1 μmol/L), but not its inactive analog, KN-92, decreased I_{Ca, L} amplitude from 12±2 to 6±1 pA/pF without altering the shape of the current-voltage relationship. Both AIP and KN-93 shifted the midpoint of the steady-state inactivation curve leftward and markedly slowed the recovery of I_{Ca, L} from inactivation. Similar results were observed using the fast Ca²⁺ chelator BAPTA, whereas the slow Ca²⁺ chelator EGTA had no significant effect, which suggests that CaMKII activity is preferentially regulated by local Ca²⁺ transients. Indeed, confocal immunocytochemical imaging showed that active CaMKII is highly localized beneath the surface membrane in the vicinity of L-type channels and that AIP and KN-93 significantly reduced CaMKII activity. Thus, we conclude that CaMKII plays a vital role in regulating cardiac pacemaker activity mainly via modulating I_{Ca, L} inactivation and reactivation, and local Ca²⁺ is critically involved in these processes.