The nature of EDHF is unknown but several pathways are likely to contribute. Potassium ion efflux from endothelial cells may induce Na,K-ATPase-dependent smooth muscle cells (SMCs) hyperpolarization and/or result in SMC hyperpolarization by passing current through myoendothelial gap junctions. We have previously developed a technique for siRNA-based downregulation of α2 isoform Na,K-ATPase in vivo in rat mesenteric small arteries and demonstrated that the specific downregulation of α2 isoform inhibited intercellular communication. We aimed to test the significance of the α2 isoform of the Na,K-ATPase for the EDHF response. We transfected rat mesenteric small arteries with α2 isoform siRNA and with eGFP-siRNA, as control. Arteries were tested under isometric conditions in vitro 3 days after transfection, where the α2 isoform Na,K-ATPase protein expression reduced to 56 ± 7% (n=5). There was no difference in acetylcholine-induced relaxation between α2 isoform downregulated and control arteries under control conditions (maximum relaxations 71 ± 10 and 80 ± 15 %, n=7, respectively). The downregulated arteries had significantly reduced EDHF-like relaxation after NO- and prostanoids-pathways inhibition with L-NAME and indomethacin (37 ± 11 and 79 ± 12 %, P < 0.05). The relaxation was completely inhibited by inhibitors of EDHF-like signal, TRAM34 and apamin. These results directly demonstrate the importance of the α2 isoform Na,K-ATPase for EDHF signalling either as an acceptor of potassium ion signal for SMC hyperpolarization, or as a modulator of myoendothelial communication.