Epithelial endometrial cells were isolated from normal and ovariectomized adult rats. The procedure involves successively (1) perfusion of the uterine vasculature with calciumfree Hanks’ solution, (2) perfusion with 0.05% collagenase and 0.1% hyaluronidase in calciumfree Hanks’ solution, (3) scraping with a rubber instrument, (4) suspension of detached cells in Eagle’s MEM and plating in small plastic containers. 95% of freshly isolated cells show typical epithelial morphology. After the 1st day of incubation, epithelial cells present two different forms, flat polygonal cells with dispersed chromatin in the nuclei and large nucleoli, and polyhedral cells grouped in spherical masses whose nuclei are denser and the nucleoli smaller. Both forms possess the typical cytology of epithelial cells in situ. At the 4th day of incubation, mitosis occurs frequently in flat cells, whereas the masses disappear progressively. Proliferating flat cells were cultivated for 15 days without signs of degeneration. Both forms of cells remain metabolically very active as demonstrated by increased 3H-uridine incorporation in response to estradiol-17