The recent discovery of a new isoform of estrogen receptor (ER)β has prompted the reexamination of estrogen action on target organs. Here, we describe the endometrial expression of human ERβ and compare its distribution with that of ERα in the endometrial functional zone. Using immunocytochemistry with well characterized polyclonal antibodies against ERβ, we have detected specific ERβ expression in all endometrial compartments (glandular, stromal, and vascular); the specificity of the immunostaining is confirmed by lack of staining of the uterine sections with anti-ERβ antibodies previously incubated with peptide preparation. The highest levels of ERβ expression are observed in epithelial cells during the periovulatory period (days 14 and 15), as well as in stromal cells and cells of the vascular wall in the late-secretory phase; both smooth muscle cells and endothelial cells express ERβ, as deduced from immunocytochemistry and RT-PCR analysis. ERβ staining is usually low compared with that of ERα, except at days 24–26. The presence of ERβ in decidualized stromal cells is deduced from immunocytochemistry using antismooth α-actin and anti-ERβ antibodies or from RT-PCR analysis of ERβ and insulin-like growth factor-BP transcripts in the same cells; the presence of ERβ-positive stromal cells located close to vascular smooth muscle cells during this period suggests some specific role of this receptor during decidualization. ERα is also present in the cells of the endometrial vascular wall, in addition to the nuclei of glandular epithelial and stromal cells. Vascular ERα expression is highest during the periovulatory period, suggesting a regulation by estradiol, and a role in vascular function. Moreover, different variations of ERβ and ERα in arterioles might have implications for the modulation of vascular function, possibly of vascular tone, during the menstrual cycle. Finally, these data suggest that ERβ may have important roles in endometrial function, in addition to the well known role of ERα in endometrial proliferation and differentiation.