The coculture of endometrial epithelial cells (EEC) with stromal cells (ESC) allows achievement of an improved in vitro system for studying interactions between cells via soluble signals. The purpose of this study was to investigate whether 17β-estradiol and insulin can induce proliferation of EEC through ESC-secreted factors. No evidence of estrogen-induced EEC proliferation has been reported so far in the conventional culture methods. To this end, we used an in vitro bicameral coculture model where human EEC were grown on extracellular matrix-coated inserts applied in dishes containing ESC. Proliferation was assessed by tritiated thymidine incorporation. Homogeneity of endometrial cell populations was ascertained immunocytochemically. 17β-Estradiol did not induce any proliferative effect on EEC cultured alone. Endometrial epithelial cell proliferation was significantly enhanced in EEC/ESC cocultures; moreover, it was further increased by 17β-estradiol addition. Insulin increased proliferation in EEC cultured alone, but again the effect was more pronounced in EEC/ESC cocultures. Coincubation of 17β-estradiol and an antibody against insulin-like growth factor I (IGF I) led to neutralization of ESC-mediated EEC proliferation. This work provides evidence that the effect of 17β-estradiol on human EEC proliferation may be mediated at least in part through ESC-secreted IGF I. We also showed that insulin effect is also partially due to ESC activation.