Voltage-gated potassium (K_v) channels in vascular smooth muscle cells (VSMC) are critical regulators of membrane potential and vascular tone. These channels exert a hyperpolarizing influence to counteract the depolarizing effects of intraluminal pressure and vasoconstrictors. However, the contribution of K_v channel activity to the functional regulation of cerebral (parenchymal) arterioles within the brain is not known. Thus K_v channel properties in parenchymal arteriolar SMCs were characterized. Isolated, pressurized parenchymal arterioles and arterioles in cortical brain slices exhibited robust constriction in the presence of the K_v channel inhibitor 4-aminopyridine (4-AP). 4-AP also decreased the amplitude of K_v currents recorded from SMCs. The steady-state activation and inactivation properties of K_v currents suggested that these channels are composed of K_v1.2 and 1.5 subunits, which was confirmed by RT-PCR. K_v channels can be regulated by extracellular glucose, which may be involved in the functional hyperemic response in the brain. Thus the effects of glucose on K_v channel activity and arteriolar function were investigated. Elevation of glucose from 4 to 14 mM significantly decreased the peak K_v current amplitude and constricted arterioles. Arteriolar constriction was prevented by inhibition of protein kinase C (PKC), consistent with previous studies showing enhanced PKC activity in the presence of elevated glucose. In cortical brain slices, the dilation generated by neuronal activity induced by electrical field stimulation was decreased by 54% in 14 mM glucose when compared with the dilation in 4 mM glucose. In anesthetized mice the whisker stimulation-induced increase in local cerebral blood flow was also significantly decreased in 14 mM glucose, and this effect was similarly prevented by PKC inhibition. These findings point to a critical role for K_v channels in the regulation of intracerebral arteriolar function and suggest that changes in perivascular glucose levels could directly alter vascular diameter resulting in a modulation of local cerebral blood flow.