We have investigated the physiological role of the “rapidly activating” delayed rectifier K+ current ( IKr) in pacemaker activity in isolated sinoatrial node (SAN) myocytes and the expression of mouse ether-a-go-go (mERG) genes in the adult mouse SAN. In isolated, voltage-clamped SAN cells, outward currents evoked by depolarizing steps (greater than –40 mV) were strongly inhibited by the class III methanesulfonanilide compound E-4031 (1–2.5 μM), and the deactivation “tail” currents that occurred during repolarization to a membrane potential of –45 mV were completely blocked. E-4031-sensitive currents ( IKr) reached a maximum at a membrane potential of –10 mV and showed pronounced inward rectification at more-positive membrane potentials. Activation of IKr occurred at –40 to 0 mV, with half-activation at about –24 mV. The contribution of IKr to action potential repolarization and diastolic depolarization was estimated by determining the E-4031-sensitive current evoked during voltage clamp with a simulated mouse SAN action potential. IKr reached its peak value (∼0.6 pA/pF) near –25 mV, close to the midpoint of the repolarization phase of the simulated action potential, and deactivated almost completely during the diastolic interval. E-4031 (1 μM) slowed the spontaneous pacing rate of Langendorff-perfused, isolated adult mouse hearts by an average of 36.5% ( n = 5). Expression of mRNA corresponding to three isoforms coded by the mouse ERG1 gene (mERG1), mERG1a, mERG1a′, and mERG1b, was consistently found in the SAN. Our data provide the first detailed characterization of IKr in adult mouse SAN cells, demonstrate that this current plays an important role in pacemaker activity, and indicate that multiple isoforms of mERG1 can contribute to native SAN IKr.