Abstract—Although Purkinje fibers (PFs) play an important role in cardiac electrophysiology, almost nothing is known about the expression of ion-channel subunits in PFs. We applied competitive reverse transcription–polymerase chain reaction, Western blotting, and immunocytochemistry to compare the expression of ion-channel subunit mRNA and protein in canine PFs versus ventricular muscle (VM). For transient outward current–related subunits, Kv4.2 was not detected, and Kv1.4 expression was extremely low. Kv4.3 expression was of the same order for VM and PFs. The tetraethylammonium chloride–sensitive subunit Kv3.4 was expressed much more strongly in PFs than in VM, and Kv channel–interacting protein transcript expression was 25-fold stronger in VM than in PFs. For delayed rectifiers, ERG and KvLQT1 expression was lower in PFs at both mRNA and protein levels. Although minK transcripts were more numerous in PFs, minK protein was significantly more strongly expressed in VM. L-type Ca2+current &agr;-subunit (CaV1.2) and Na+-Ca2+exchanger proteins were more strongly expressed in VM than in PFs. For T-type Ca2+current, CaV3.1, CaV3.2, and CaV3.3 transcripts were all more strongly expressed in PFs. For the nonselective cation current, hyperpolarization-activated cation channel 1 (HCN1) expression was subquantifiable, HCN2 transcript expression was comparable in PFs and VM, and HCN4 mRNA expression was strong in PFs but below the detection threshold in VM. HCN2 and HCN4 protein expression was much stronger in PFs than in VM. We conclude that ion-channel subunit expression in PFs differs from that in VM in ways that are consistent with, and shed light on the molecular basis of, well-recognized fundamental PF ionic properties.