KCNQ4-encoded voltage-dependent potassium channels (Kv7.4) are important regulators of vascular tone and are severely compromised in models of hypertension (1, 2). Moreover, we have recently demonstrated that these ion channels underlie β-adrenoceptor-stimulated relaxation in rat renal arteries (2). The present study investigates the contribution of Kv7.4 channels to cAMP-mediated relaxation in mesenteric and middle cerebral arteries and there function in spontaneously hypertensive rats (SHRs). Third-order branches of the superior mesenteric artery and middle cerebral arteries were isolated from male Wistar rats or SHRs (200-225 g) in cold Krebs’ solution. Arteries were mounted in a wire myograph and placed under a tension equivalent to that generated at 0.9 times the diameter of the vessel at 100 mmHg (mesenteric arteries) or 70 mmHg (middle cerebral arteries). Responses to isoprenaline or calcitonin gene-related peptide (CGRP) were determined in the absence and presence of the Kv7 channel blocker linopirdine. Some experiments were carried out in arteries depleted of KCNQ4 by transfecting them with selective siRNA using either reverse permeabilization or TransIT-LT transfection reagent (Mirus). Relaxation of mesenteric arteries by isoprenaline and middle cerebral arteries by CGRP was inhibited by 10 µM linopirdine (~30% and 70% attenuation in Emax, respectively). Mesenteric and middle cerebral arteries incubated with siRNA targeted against KCNQ4 were less responsive to the Kv7 activator S-1 compared to vessels incubated with scrambled siRNA. Relaxation in response to isoprenaline (mesenteric artery) and CGRP (middle cerebral artery) were significantly reduced (1-log unit rightward shift in pEC50) in vessels depleted of KCNQ4. In mesenteric arteries from SHRs, the relaxation to S-1 was drastically impaired coinciding with a marked attenuation in responses to isoprenaline. Conversely, S-1 and CGRP induced relaxations were not different between middle cerebral arteries from SHRs and normotensive rats. This study demonstrates that Kv7.4 channels contribute to the relaxation of mesenteric and middle cerebral arteries associated with activation of receptors coupled to adenylyl cyclase (β-adrenoceptors and CGRP-like receptors, respectively). In addition, the present study indicates that whilst Kv7.4 channel activity is drastically impaired in mesenteric, aorta (1) and renal (2) arteries of SHRs, middle cerebral arteries are unaffected. Further studies are required to ascertain whether Kv7.4 channels are protected in the cerebral vasculature of SHRs or if other Kv7 subtypes compensate for their function.