Background and Aim: The action potential plateau of Purkinje fibers is particularly sensitive to tetrodotoxin (TTX) and this could be due to a TXX-sensitive Na+ current. The expression of TTX-sensitive neuronal Nav1.1 and Nav1.2 isoforms has been reported in canine Purkinje myocytes. Our aim was to investigate by means of biochemical and functional techniques whether the TTX-sensitive skeletal Nav1.4 isoform is also expressed in canine cardiac Purkinje myocytes. Methods and Results: Using Nav1.4 specific primers, a PCR product corresponding to Nav1.4 was amplified from canine Purkinje fibers RNA and confirmed by sequencing and megablast of the gene bank. Confocal indirect immunostaining using anti-Nav1.4 antibody demonstrates distinct sarcolemmal staining pattern compared to that of the cardiac isoform Nav1.5. Expression of Nav1.4 in tsA201 cells yielded a TTX-sensitive Na+ current with an IC50 of 10 nM. Conclusions: These results demonstrate the expression of the TTX-sensitive Nav1.4 channel in canine cardiac Purkinje myocytes. This novel finding suggests a role of Nav1.4 channel in Purkinje myocytes and thus has important clinical implications for the mechanisms and management of ventricular arrhythmias originating in the Purkinje network. Published by Elsevier Inc.