The goal of this study was to determine how myogenic responses and vascular responses to reduced PO₂ interact to determine vascular smooth muscle (VSM) transmembrane potential and active tone in isolated middle cerebral arteries from Sprague-Dawley rats. Stepwise elevation of transmural pressure led to depolarization of the VSM cells and myogenic constriction, and reduction of the O₂ concentration of the perfusion and superfusion reservoirs from 21% O₂ to 0% O₂ caused vasodilation and VSM hyperpolarization. Myogenic constriction and VSM depolarization in response to transmural pressure elevation still occurred at reduced PO₂. Arterial dilation in response to reduced PO₂ was not impaired by pressure elevation but was significantly reduced at the lowest transmural pressure (60 mmHg). However, the magnitude of VSM hyperpolarization was unaffected by transmural pressure elevation. This study demonstrates that myogenic activation in response to transmural pressure elevation does not override hypoxic relaxation of middle cerebral arteries and that myogenic responses and hypoxic relaxation can independently regulate vessel diameter despite substantial changes in the other variable.