The purpose of this study was to determine the role of estrogen in neuropeptide Y (NPY) and Y(1) receptor (Y(1)R)-mediated vascular responses in female rats. Based on earlier work from our laboratory that female rats lacked an NPY contribution to hindlimb vascular conductance relative to males, we tested the hypothesis that estrogen modulates Y(1)R-mediated hindlimb blood flow control. Thus it was expected that ovariectomy would: 1) increase skeletal muscle Y(1)R expression, 2) decrease skeletal muscle Y(2) receptor (Y(2)R) expression, 3) decrease peptidase activity, and/or 4) increase overall skeletal muscle NPY concentration. Separate groups of control (CTL), ovariectomized (OVX), and OVX + 17beta-estradiol replacement (OVX + E(2); 21-day pellet) rats were studied. Animals were anesthetized and given localized hindlimb delivery of BIBP-3226 (Y(1)R antagonist), while femoral artery blood flow and blood pressure were recorded. Tissue samples from the white and red vastus lateralis muscle were extracted to examine Y(1)R and Y(2)R expression, peptidase activity, and NPY concentration. We found that Y(1)R blockade resulted in increased baseline hindlimb blood flow and vascular conductance in OVX rats, whereas no change was noted in CTL or OVX + E(2) groups (P < 0.05). This enhanced functional effect in the OVX group aligned with greater skeletal muscle Y(1)R expression in white vastus muscle and a substantial increase in NPY concentration in both white and red vastus muscle compared with CTL and OVX + E(2) groups. There was no change in Y(2)R expression or peptidase activity among the groups. These data support the hypothesis that estrogen blunts Y(1)R activation in the rat hindlimb through an effect on Y(1)R expression and NPY concentration.