Objective: To evaluate the phenotype, proliferative, and differentiation capacities in vitro of stromal cells derived from peritoneal, ovarian, and deeply infiltrating endometriosis. Design: Experimental study using phase contrast microscopy, immunocytochemistry, and functional bioassays. Setting: University- based laboratory. Patient(s): Women with and without endometriosis undergoing surgery for benign indications. Intervention( s): None. Main Outcome Measure(s): The stability in vitro of stromal cells derived from peritoneal (n = 18), ovarian (n = 29), and deeply infiltrating (n = 14) endometriotic lesions, as well as endometrium from women with (n = 5) and without endometriosis (n = 5)was evaluated by detection of endometrial markers. The proliferative and differentiation capacity of the cells was assessed by the use of cell doubling estimation and in vitro decidualization assays. Result(s): The expression of the progesterone receptor and CD10 in stromal cells derived from the three types of endometriotic lesions is retained in culture up to passage 10. The doubling time of stromal cells from deeply infiltrating lesions is lower than that of endometrial stromal cells. Levels of prolactin and insulin- like growth factor binding protein- 1 (IGFBP- 1) are reduced in supernatants from stromal cells derived from the three types of lesions and from the endometrium of women with endometriosis. Conclusion(s): The peritoneal, ovarian, and deeply infiltrating endometriotic stromal cell lines we describe retain in vivo tissue markers. Loss of differentiation capacity of the endometriotic cell lines and endometrial cells from women with endometriosis may influence the capacity for proliferation and survival of these cells in the ectopic environment. Objective: To evaluate the phenotype, proliferative, and differentiation capacitation in vitro of stromal cells derived from peritoneal, ovarian, and deeply infiltrating endometriosis. Design: Experimental study using phase contrast microscopy, immunocytochemistry, and functional bioassays. Intervention (s): None. Main Outcome Measure (s): The stability in vitro of stromal cells derived from peritoneal (n = 18), ovarian (n = 29), and deeply infiltrating (n = 14) endometriotic lesions, as well as endometrium from women with (n = 5) and without endometriosis (n = 5) was evaluated by detection of endometrial markers. The proliferative and differentiation capacity of the cells was assessed by the use of cell doubling estimation and in vitro decidualization assays. (s): The expression of the progesterone receptor and CD10 in stromal cells derived from the thre e types of endometriotic lesions are retained in culture up to passage 10. The doubling time of stromal cells from deeply infiltrating lesions is lower than that of endometrial stromal cells. Levels of prolactin and insulin- like growth factor binding protein- 1 Conclusion (s): The peritoneal, ovarian, and deeply infiltrating endometriotic stromal cell lines we describe retained in vivo tissue markers. Loss of differentiation capacity of the endometriotic cell lines and endometrial cells from women with endometriosis may influence the capacity for proliferation and survival of these cells in the ectopic environment.