The multifunctional Ca⁺⁺/calmodulin-dependent protein kinase II (CaM kinase) mediates Ca⁺⁺-induced augmentation of L-type Ca⁺⁺ current (I_Ca); therefore it may act as a proarrhythmic signaling molecule during early afterdepolarizations (EADs) due to I_Ca. To investigate the hypothesis that I_Ca-dependent EADs are favored by CaM kinase activation EADs were induced with clofilium in isolated rabbit hearts. All EADs were rapidly terminated with I_Caantagonists. Hearts were pretreated with the CaM kinase inhibitor KN-93 or the inactive analog KN-92 (0.5 μM) for 10 min before clofilium exposure. EADs were significantly suppressed by KN-93 (EADs present in 4/10 hearts) compared to KN-92 (EADs present in 10/11 hearts) (P = .024). There were no significant differences in parameters favoring EADs such as monophasic action potential duration or heart rate in KN-93- or KN-92-treated hearts. CaM kinase activity in situincreased 37% in hearts with EADs compared to hearts without EADs (P = .015). This increase in CaM kinase activity was prevented by pretreatment with KN-93. In vitro, KN-93 potently inhibited rabbit myocardial CaM kinase activity (calculatedK_i ≤ 2.58 μM), but the inactive analog KN-92 did not (K_i > 100 μM). The actions of KN-93 and KN-92 on I_Ca and other repolarizing K⁺currents did not explain preferential EAD suppression by KN-93. These data show a novel association between CaM kinase activation and EADs and are consistent with the hypothesis that the I_Ca and CaM kinase activation both contribute to EADs in this model.