This study examined the role of the actin cytoskeleton in Rho-kinase-mediated suppression of the delayed rectifier K⁺ (K_DR) current in cerebral arteries. Myocytes from rat cerebral arteries were enzymatically isolated and whole cell K_DR currents monitored using conventional patch clamp electrophysiology. At +40 mV, the K_DR current averaged 19.8 ± 1.6 pA/pF (mean ± SE) and was potently inhibited by uridine triphosphate (UTP; 3 x 10^-5 M). This suppression was observed to depend on Rho-signaling and was abolished by the Rho-kinase inhibitors H-1152 (3 x 10^-7 M) and Y-27632 (3 x 10^-5 M). Rho-kinase was also found to concomitantly facilitate actin polymerization in response to UTP. We therefore examined whether actin dynamics played a role in the ability of Rho-kinase to suppress K_DR current and found that actin disruption using either cytochalasin D (1 x 10^-5 M) and latrunculin A (1 x 10^-8 M) prevented current modulation. Consistent with our electrophysiological observations, both Rho-kinase inhibition and actin disruption significantly attenuated UTP-induced depolarization and constriction of cerebral arteries. We propose that UTP initiates Rho-kinase-mediated remodeling of the actin cytoskeleton and consequently suppresses the K_DR current, thereby facilitating the depolarization and constriction of cerebral arteries.