Existing evidence suggests that neuropeptide Y (NPY) acts as a neurotransmitter in vascular smooth muscle and is co-released with norepinephrine from sympathetic nerves. We hypothesized that release of NPY stimulates NPY Y₁ receptors in the skeletal muscle vasculature to produce vasoconstriction during dynamic exercise. Eleven mongrel dogs were instrumented chronically with flowprobes on the external iliac arteries of both hindlimbs and a catheter in one femoral artery. In resting dogs (n=4), a 2.5 mg bolus of BIBP 3226 (NPY Y₁ antagonist) infused into the femoral artery increased external iliac conductance by 150±82% (1.80±0.44 to 3.50 ±0.14 ml*min^-1*mmHg^-1, p<0.05). A 10 mg bolus of BIBP 3226 infused into the femoral artery in dogs (n=7) exercising on a treadmill at a moderate intensity (6 miles*h^-1) increased external iliac conductance by 28±6% (6.00±0.49 to 7.64±0.61, ml*min^-1*mmHg^-1, p<0.05) while the solvent vehicle did not (5.74±0.51 to 5.98±0.43, ml*min^-1*mmHg^-1, p>0.05). During exercise BIBP 3226 abolished the reduction in conductance produced by infusions of the NPY Y₁ agonist, (Leu³¹, Pro³⁴)-neuropeptide Y (-19±3% vs. 0.5±1%). Infusions of BIBP 3226 (n=7) after alpha adrenergic receptor antagonism with prazosin and rauwolscine also increased external iliac conductance 6.82±0.43 to 8.22±0.48, ml*min^-1*mmHg^-1, p<0.05. These data support the hypothesis that NPY Y₁ receptors produce vasoconstriction in exercising skeletal muscle. Furthermore, the NPY Y₁ receptor mediated tone appears to be independent of alpha adrenergic receptor mediated vasoconstriction.