During ovine pregnancy, when both estrogen and progesterone are elevated, prostacyclin (PGI₂) production by uterine arteries and the key enzymes for PGI₂ production, phospholipase A₂ (cPLA₂), cyclooxygenase 1 (COX-1), and prostacyclin synthetase (PGIS), are increased. This study was conducted to determine whether exogenous estradiol-17β (E₂β) with or without progesterone (P₄) treatment would increase cPLA₂, COX-1, and PGIS protein expression in ovine uterine, mammary, and systemic (renal, mental, and coronary) arteries. Nonpregnant ovariectomized sheep received vehicle (n = 10), P₄ (0.9-g controlled internal drug release vaginal implants; n = 13), E₂β (5 μg/kg bolus followed by 6 μg kg⁻¹ day⁻¹; n = 10), or P₄ E₂β (n = 12). Arteries were procured on Day 10, and cPLA₂, COX-1, and PGIS protein were measured by Western immunoblot analysis in endothelial isolated proteins and vascular smooth muscle (VSM). The levels of cPLA₂ was increased in uterine artery endothelium in ewes treated with P₄ E₂β but was not altered by any steroid treatment in renal, coronary, mammary, or omental artery endothelium or in VSM of any evaluated artery. Similarly, COX-1 was increased in uterine artery endothelium with P₄ E₂β but was not significantly altered by treatment in other endothelium or VSM. E₂β treatment increased PGIS protein in uterine and renal artery endothelium but did not alter PGIS in other endothelial tissue. P₄ increased PGIS expression in the uterine, mammary, omental, and renal artery VSM, and E₂β increased PGIS expression in the uterine and omental artery VSM. Both E₂β and P₄ treatments differentially alter protein expression of the key enzymes involved in PGI₂ production in different artery types and may play an important role in the control of blood flow redistribution during hormone replacement therapy.