Despite the key role of Kv7.4 channels in the regulation of vascular tone, and their involvement in mediating Gs-coupled vasodilator responses [1], the factors that regulate channel activity are poorly understood. Moreover, the signals linking Kv7.4 to receptor stimulation is still unclear. In this work we studied possible interactions between Kv7.4 channels and Gβγ subunits in β-adrenoreceptor mediated responses in rat renal arteries (RRA). Membrane currents were measured using cell-attached configuration of patch-clamp technique on single myocytes freshly isolated from RRA. The cells were patched with a cocktail of K+ channel blockers in the pipette and generated small amplitude (≈0.2 pA) currents with apparent NPo 0.025±0.009 (n=7, mean±SEM) when the cell membrane was depolarised to 0 mV. Isoproterenol (ISO, 1 µM) applied to the bath solution increased the NPo to 0.102±0.023 (n=7, p<0.01, unpaired Student’s t-test). The presence of pertussis toxin did not affect basal channel activity (0.026±0.012, n=6) and ISO induced a small increase of NPo to 0.055±0.033 (n=5), this was not statistically significant (p=0.437). Linopirdine (10 µM), a pan-Kv7 blocker, in a patch pipette abolished the single channel activity, but did not produce significant changes if applied to the bath solution (n=4). In the presence of gallein (100 µM), a molecular inhibitor of Gβγ activity, basal NPo was negligible, and the stimulatory effect of ISO was prevented (NPo 0.002±0.001, n=5). In HEK293 cells stably expressing Kv7.4 channels addition of 2ng/ml Gβγ subunits to inside-out patches increased NPo from 0.035±0.006 (n=14) to 0.117±0.034 (n=3, p<0.05), and 50ng/ml Gβγ subunits increased it further to 0.343±0.062 (n=6, p<0.001). Single channel conductance of these Kv7.4 channels was calculated as 2.31 pS, which is similar to the native linopirdine-sensitive channels in RRA myocytes. The functional role of Gβγ subunits on the activity of Kv7 channels was checked on RRA segments using isometric tension myography. Both linopirdine and gallein produce a similar robust contraction. Linopirdine did not produce any further contraction if added after gallein consistent with a role for Gβγ subunits dictating Kv7 activity. 10μM mSIRK, a cell-permeable Gβγ binding peptide, which causes disassociation of Gβγ subunits from α subunits without stimulating nucleotide exchange [2] relaxed pre-contracted renal arteries by 36.1±2.8% (n=7) that was prevented by linopirdine (13.1±2.2%, n=7) or gallein (13.5±5.5%, n=6). Similarly, ISO relaxations of RRA were sensitive to Kv7 blockade and gallein as well significantly inhibited ISO-mediated relaxations of RRA. Our study suggest the view that Gβγ subunit interaction is obligatory for effective function of vascular Kv7 channels and when compromised can lead to diminished ISO-mediated vasorelaxation.