[Abstract] The isolation and primary culture of cells from human endometrial biopsies provides valuable experimental material for reproductive and gynaecological research. Whole endometrial biopsies are collected from consenting women and digested with collagenase and DNase I to dissociate cells from the extracellular matrix. Cell populations are then isolated through culturing, filtering and magnetic separation using cell-surface antigen markers. Here we provide a comprehensive protocol on how to isolate and culture individual cell types from whole endometrial tissues for use in in vitro experiments.[Background] The human endometrium is the inner most mucosal layer of the uterus. It consists of a columnar epithelium and basal stromal layer that undergoes cyclical regeneration, growth and transformation in response to circulating hormones. The differentiation of the endometrial lining into a glandular secretory phenotype provides a hospitable environment for blastocyst implantation and successful pregnancy. In the absence of pregnancy this layer is shed, leading to menstruation. The isolation and culture of cells from human endometrial biopsies allows for in vitro functional assessment and the study of cell characteristics in relation to patient outcomes. The isolation and culture of endometrial cells is an invaluable research model to investigate many aspects of gynaecological and obstetrical medicine including infertility, implantation failure, recurrent miscarriage and menstrual disorders. Whole human endometrial biopsies contain human endometrial stromal cells (HESCs), luminal and glandular endometrial epithelial cells (HEECs), red blood cells and a mixed population of immune cells. HESCs can be easily and inexpensively isolated from whole biopsies and actively proliferate in culture for up to 5 passages without significant change in their growth dynamics. This provides a large window of opportunity for experimental analysis. Furthermore, within dissociated HESCs there is a sub-population of perivascular progenitor mesenchymal stem-like cells that can be isolated using the perivascular-specific antigen SUSD2 and its cognate antibody W5C5. Here we provide in detail an updated and expanded protocol from those published previously (Masuda et al., 2012;Chen and Roan, 2015) to describe steps in isolating and culturing different cell types from whole human endometrium. We provide further information on biopsy collection, detailed protocols for isolation of progenitor cells and additional procedures to increase epithelial cell yield and culturing efficiency. Materials and Reagents