Atrial fibrillation (AF) is a fast and irregular activation of the atria that represents the most common clinical arrhythmia. AF is associated with significant mortality and morbidity. While sodium channel blockers can be used as part of a rhythm-control strategy, their use is limited due to the risk of potentially lethal ventricular tachyarrhythmias in patients with cardiac structural abnormalities (Hindricks et al., 2020). It has been suggested that atrial-ventricular differences in Na+ currents (INa) may be exploited for atrial selective antiarrhythmic drug action without risk of ventricular side-effects (Hancox et al., 2016). Eleclazine (GS-6615) is a putative anti-arrhythmic drug that has been shown to be safe and well-tolerated in patients and have properties similar to the prototypical atrial-selective Na+ channel blocker, ranolazine (Caves et al., 2017; El-Bizri et al., 2018). At a previous meeting, we presented data from rat isolated cardiac myocytes showing atrial-ventricular differences in INa and suggested an atrial-selective action of eleclazine, although the inhibitory mechanism was unclear (Caves et al., 2019). The present study investigated the mechanisms of INa inhibition by eleclazine in left atrial and left ventricular myocytes isolated from adult male Wistar rat hearts. Procedures were approved by local ethics committee and performed in accordance with UK legislation.  INa was recorded at room temperature (~22 °C) using whole-cell patch clamp recording techniques with symmetrical external and internal [Na+] (5 mM).  The use-dependence of INa inhibition by eleclazine (10 µM) was examined at holding potentials (HP) of -120 mV and -100 mV using a series of 40 pulses of 20 ms duration to -30 mV at diastolic intervals (DI) of 40 ms and 110 ms. Data are presented as mean ± standard error of the mean, compared by unpaired t-test and the limit of statistical confidence is P<0.05. Eleclazine caused use-dependent inhibition of INa in both atrial (HP=-120 mV, n=12; HP=-100 mV, n=8) and ventricular (HP=-120 mV, n=9; HP=-100 mV, n=9) myocytes that was greater at DI=40 ms (HP=-120 mV: atrial 37.5±2.6%; ventricular 36.2±5.1%; HP=-100 mV: atrial 22.9±6.3%; ventricular 23.8±4.1%) than DI=110 ms (HP=-120 mV: atrial 17.1±1.9%; ventricular 15.7±3.2%; HP=-100 mV: atrial 8.6±2.0%; ventricular 10.4±1.9%). However, there was no difference between the two cell-types in the use-dependent inhibition of INa at either HP. Eleclazine also produced an instantaneous inhibition of INa, which was greater at HP=-100 mV than at HP=-120 mV in both cell types. Notably, at HP=-120 mV, the instantaneous inhibition was greater in atrial (9.1±1.9%) than in ventricular myocytes (-0.8±2.2%; P=0.0031) whereas at HP=-100 mV there was no difference between the two cell-types (atrial: 28.8±6.3%; ventricular: 29.1±4.7%). Eleclazine caused a negative shift in the voltage of half-maximal inactivation and slowed the recovery of INa from inactivation in both cell types. Increasing the duration of the test pulse to 200 ms had no effect on eleclazine inhibition in either cell type. In summary, the data are consistent with preferential activated state block of Na+ channels by eleclazine in atrial and ventricular myocytes (Caves et al., 2020). Eleclazine warrants further investigation as an atrial-selective anti-arrhythmic drug.