Estrogen is recognized to be one of the driving forces in increases in uterine blood flow through both rapid and delayed actions via binding to its receptors, ERα and ERβ at the uterine artery (UA) wall, and especially in UA endothelium (UAE). However, information regarding estrogen receptor (ER) expression in UAE is limited. This study was designed to test whether ERs are expressed in UAE in vivo, and if they are, whether these receptors are maintained in cultured UA endothelial cells (UAECs) in vitro. By using immunohistochemical and Western blot analyses, we clearly demonstrated ERα and ERβ protein expression in pregnant (Days 120–130) sheep UA and UAE in vivo and as well as cultured UAECs in vitro. Reverse transcription-polymerase chain reaction (RT-PCR) amplified both ERα and ERβ mRNAs in UA, UAE, and UAECs. Of interest, a truncated ERβ (ERβ2) variant due to a splicing deletion of exon 5 of the ERβ gene was detected in these cells. Quantitative RT-PCR analysis revealed that ERα mRNA levels are ∼8-fold (P < 0.01) higher than that of ERβ in UAECs, indicating that ERα may play a more important role than ERβ in the UAEC responses to estrogen. Fluorescence immunolabeling analysis showed that ERα is present in both nuclei and plasma membranes in UAECs, and the latter is also colocalized with caveolin-1. The membrane and nuclear ERα presumably participate in rapid and delayed responses, respectively, to estrogen on UAE. Taken together, our data demonstrated that UAE is a direct target of estrogen actions and that the UAEC culture model we established is suitable for dissecting estrogen actions on UAE.