Neuropeptide Y (NPY) is a peptide transmitter released from sympathetic neurons, promoting potent and prolonged vasoconstriction. We recently reported that male (1), but not female, rats exhibited baseline endogenous NPY Y1-receptor (Y1R) modulation of hindlimb vasculature (2). The lack of Y1R control in females was evident despite Y1R expression and NPY in the hindlimb. Subsequently, we observed that female rats limit NPY bioavailability via activation of inhibitory NPY Y2-receptors (Y2R), greater skeletal muscle Y2R expression and NPY metabolism via augmented peptidase activity (3). In this investigation we sought to determine the underlying mechanism(s) governing the sexual dimorphism in NPY control of skeletal muscle vasculature. We tested the hypothesis that estrogen minimizes NPY bioavailability and/or Y1R vasomotor control in skeletal muscle. Thus, we examined whether ovariectomy would expose an important contribution of endogenous Y1R activation to baseline blood flow in female rats as a result of 1) increased skeletal muscle Y1R expression, 2) increased skeletal muscle NPY, and 3) decreased peptidase activity. We further assessed whether estrogen replacement would reverse the impact of ovariectomy on cellular and functional vascular responses. In terminal experiments, animals were anaesthetized by intraperitoneal injection of α-chloralose (80 mg/kg) and urethane (500 mg/kg). Animals were killed by anaesthetic overdose. Recovery surgeries (i.e. ovariectomy and hormone pellet insertion) were carried out under pentobarbital anaesthesia. In ovariectomized rats treated with estradiol placebo (OVX, n=7), localized hindlimb arterial infusion of the Y1R antagonist BIBP3226 (100 µg/kg) increased blood flow (Δ from baseline = 270.2±74 µl/min) and vascular conductance (Δ from baseline = 2.96±0.95 µl/min/mmHg) (mean ± SEM, one-way ANOVA, P<0.05). In contrast, Y1R blockade had no vascular effect on the control group (CTRL, n=5) or on ovariectomized rats treated with 17β-estradiol (OVX+E2, n=6). The OVX group had augmented Y1R expression in white vastus muscle (WV) (one-way ANOVA, P<0.05, Western blot, n=8 per group) compared to the CTRL group; this effect of ovariectomy was not apparent in the OVX+E2 group (expressed in arbitrary units: CTRL = 87.6 vs. OVX = 107.4 vs. OVX+E2 = 78.9). Y1R expression was unchanged by any intervention in red vastus muscle (RV). In WV and RV, NPY concentration was elevated in OVX compared to CTRL and OVX+E2 (WV CTRL = 12±1.5 pg/μg vs. OVX = 21±2.4 pg/μg vs. OVX+E2 = 10±1 pg/μg; RV CTRL = 24±3.7 vs. OVX = 61±1.3 pg/μg vs. OVX+E2 = 31±4.7 pg/μg)(one-way ANOVA, P<0.05,ELISA, n=8 per group). Peptidase activity was unchanged among groups. Our data indicate that estrogen blunts Y1R activation in the hindlimb of baseline female rats due to an impact on Y1R expression and NPY bioavailability.