Ovariectomized (OVX) ewes were assigned to receive vehicle, progesterone (P4, 0.9-g controlled internal drug release vaginal implants), estradiol-17β (E₂, 5 μg/kg bolus 6 μg kg⁻¹ day⁻¹), or P4 E₂ for 10 days (n = 3/group). Uterine artery endothelial proteins were mechanically isolated on Day 10. The samples were used for protein expression profiling by the Ciphergen Proteinchip system and immunoblotting analysis of endothelial nitric oxide synthase (NOS3, also termed eNOS) and caveolin 1. Uterine artery rings were cut and analyzed by immunohistochemistry to localize NOS3 and caveolin 1 expression. With the use of the IMAC3 protein chip with loading as little as 2 μg protein/sample, many protein peaks could be detected. Compared to vehicle controls, a ∼133.1-kDa protein was identified to be upregulated by 2- to 4-fold in OVX ewes receiving E₂, P4, and their combination, whereas a ∼22.6-kDa protein was downregulated by 2- to 4-fold in OVX ewes receiving E₂ and E₂/P4, but not P4 treatments. Western blot analysis revealed that E₂, P4, and their combination all increased NOS3 protein, whereas E₂ and its combination with P4, but not P4 alone, downregulated caveolin 1 expression. Immunohistochemical analysis revealed that NOS3 was mainly localized in the endothelium and upregulated by E₂, whereas caveolin 1 was localized in both endothelium and smooth muscle and downregulated by E₂. Thus, our data demonstrate that uterine artery endothelial NOS3 and caveolin 1 are regulated reciprocally by estrogen replacement therapy. In keeping with the facts that E₂, but not P4, causes uterine vasodilatation and that E₂ and P4 increase NOS3 expression, but only E₂ decrease caveolin 1 expression, our current study suggests that both increased NOS3 expression and decreased caveolin 1 expression are needed to facilitate estrogen-induced uterine vasodilatation.