Coexpression of CD140b (PDGFRbeta) and CD146 has been used to isolate endometrial mesenchymal stem-like cells (eMSCs), which have a perivascular location. This study aims to evaluate a single marker for purifying eMSCs.Using an antibody panel with novel specificities, we screened human endometrial tissues and stromal cell suspensions by flow cytometry and immunohistochemistry to identify perivascular markers. Sorted subpopulations were examined for colony forming unit (CFU), self-renewal and differentiation assays for mesenchymal stem cell (MSC) function. We also transplanted sorted eMSCs under the kidney capsule of super-immunodeficient NSG mice. Magnetic bead selection was compared with flow cytometry sorting (flow-sorting) using CFU assay.One novel marker (W5C5) was particularly effective in selecting eMSCs. W5C5 cells comprise 4.2 +/- 0.6 (n=34) of endometrial stromal cells and reside predominantly in a perivascular location in both basal and functional layers of endometrium. The clonogenicity of W5C5 cells is significantly greater than W5C5 and unselected cells. W5C5 cells differentiated into adipocytes, osteocytes, chondrocytes, myocytes and endothelial cells. W5C5 cells produce endometrial stromal-like tissue in vivo. In terms of clonogenicity, magnetic bead selected W5C5 cells gave rise to significantly higher CFU numbers compared to flow-sorted W5C5 cells.This study identified W5C5 as a single marker capable of purifying eMSCs possessing MSC properties and reconstituting endometrial stromal tissues in vivo. W5C5 enriches eMSCs to high purity and provides a simple protocol for their prospective isolation using magnetic bead selection rather than flow-sorting. W5C5 selection may provide an alternate, readily available autologous source of MSC, obtainable with minimal morbidity using an office endometrial biopsy procedure for future cell-based therapies.