The late sodium current (INa,L) plays pathophysiological role in several heart diseases. Despite this, the contribution of INa,L to the cardiac ventricular action potential (AP) has not been characterized yet under physiological circumstances. We wanted to visualize INa,L during a canine ventricular AP and to determine the effect of INa,L blockade on the AP shape and on the short term variability of the AP duration (APD). Experiments were performed in isolated canine left ventricular myocytes produced by enzymatic digestion. Intramuscular application of 10 mg/kg ketamine hydrochloride and 1 mg/kg xylazine hydrochloride was used to achieve complete narcosis in adult healthy mongrel dogs of either sex as approved by the local ethical committee. We used the AP voltage clamp technique to visualize INa,L during the AP by 1 µM GS-458967 (GS). Conventional microelectrode technique was used to determine the effects of INa,L blockade on AP morphology. Under the AP, the GS sensitive current showed a peak at the AP upstroke (likely because of early sodium current (INa,E) inhibition), and then showed a sustained, gradually decreasing current due to INa,L. At 1 s pacing cycle length (PCL) GS reduced the maximal rate of depolarization (Vmax) by 44% (from 278.5±9.5 to 155.0±17.8 V/s) and AP amplitude (APA) by 15% (from 110.2±1.8 to 93.8±4.4 mV). These effects are likely due to the small INa,E blocking effect of GS. GS shortened the APD by 14% (from 209.4±5.8 to 179.6±10.9 ms) measured at 90% of repolarization and depressed the mid-plateau membrane potential by 6 mV. GS decreased short term APD variability by 18% (from 2.7±0.3 to 2.2±0.5 ms). At shorter PCL, effects of GS on APA and Vmax were more pronounced, indicating a stronger INa,E blockade under these conditions. Effects of GS on APD, mid-plateau potential and short term APD variability were less prominent with shorter PCL. In our experiments the profile of INa,L was successfully recorded under physiological conditions. The blockade of INa,L has been shown to affect AP morphology and APD short term variability. Based on these results, INa,L significantly contributes to the electrophysiological characteristics of cardiac ventricular myocytes.