Background: Unlike in peripheral vessels, the endothelium-derived hyperpolarizing factor (EDHF)-mediated component to P2Y<sub>2</sub> receptor-mediated dilations is significantly attenuated in the middle cerebral artery (MCA) of female rats compared to male rats. One aspect to the EDHF phenomenon is activation of the intermediate calcium-sensitive potassium (IK<sub>Ca</sub>) channels located on the endothelium. In an attempt to pinpoint the site along the EDHF pathway that is compromised in females, we tested the hypothesis that direct activation of IK<sub>Ca</sub> channels with DCEBIO would elicit attenuated hyperpolarization in the endothelium and smooth muscle of females compared to males. Methods: Inhibitors of nitric oxide synthase and cyclooxygenase were present throughout all experiments. Vessel diameter changes were assessed in pressurized and luminally perfused MCAs. Membrane potential changes in the endothelium and smooth muscle were measured using the perforated patch clamp method and sharp electrodes, respectively. Results and Conclusions: The maximum vasodilation to 3 × 10<sup>–4</sup> M DCEBIO was significantly reduced in females (37 ± 9%) compared to intact males (70 ± 4%). Endothelial cell hyperpolarization to DCEBIO was similar in both males and females. Smooth muscle cell hyperpolarization was attenuated in females (2 ± 1 mV) compared to males (15 ± 3 mV). Taken together, our data suggest that the transfer of hyperpolarization from the endothelium to the smooth muscle is impeded in the female rat MCA.