The slow component of the delayed rectifier K⁺ current (I _Ks) plays an important role in the repolarization of action potentials in cardiac pacemaker cells and ventricular myocytes, and is regulated by various signaling pathways. Recent evidence has shown that calmodulin (CaM) is involved in modulation of diverse ion channels in cardiac myocytes under physiological and pathophysiological conditions. In the present study, we examined regulation of I _Ks by Ca²⁺/CaM in guinea pig sinoatrial (SA) node cells using the whole-cell patch-clamp method. The density of I _Ks was larger during intracellular dialysis with a higher Ca²⁺ concentration (pCa 7, Ca (+)) compared to that with a low Ca²⁺ concentration (pCa 10, Ca (−)). Intracellular application of CaM (400 nM) markedly potentiated I _Ks with a Ca (+) pipette solution but not with a Ca (−) solution, thus showing that CaM potentiates I _Ks in an intracellular Ca²⁺-dependent manner. Intracellular application of a specific Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) inhibitor, autocamtide-2 inhibitory peptide (AIP, 500 nM), markedly reduced I _Ks activity in the presence of higher intracellular Ca²⁺. Similarly, bath application of another inhibitor, KN-93 (1 μM) also significantly suppressed I _Ks. Finally, the stimulatory action on I _Ks of Ca²⁺/CaM was abolished by pretreatment with KN-93. Taken together, these observations suggest that Ca²⁺/CaM stimulates I _Ks in guinea pig SA node cells through activation of CaMKII. This enhancement of I _Ks by CaMKII may be involved in modulation of SA node automaticity under physiological or pathophysiological condition.