OBJECTIVE In this study, we have established an in vitro three-dimensional (3D) coculture, where normal endometrial stromal cells and endometrial cancer cells were cocultured under defined hormonal conditions, to investigate the potential paracrine effects on synthesis and secretion of matrix metalloproteinases (MMPs) and, thus, cancer invasion. METHODS Endometrial stromal cells were obtained by biopsy, cultured in the presence of 100 nM estrogen for 3 days, and then mixed with extracellular matrix (ECM) composed of collagen I and matrigel in a 4:1 ratio. After 3 more days in culture, a 3D coculture was established with HEC-1A cells, an endometrial adenocarcinoma cell line, grown on top of the mixture under various ovarian steroids (no steroid, 100 nM beta-estradiol (E2), or 1.0 microM progesterone (P4)) for 10 days. The expression and localization of MMP-2, MMP-9, and integrin beta 3 subunit were visualized by immunocytochemistry and analyzed by reverse transcription polymerase chain reaction (RT-PCR). The extent of cancer invasion was quantified by Boyden's chamber assay. RESULTS Integrin beta 3 subunit was localized along the cell surface of HEC-1A cell under all three hormonal conditions. MMP-2 was located in the cytoplasm of stromal cells and on the surface of HEC-1A cells. Synthesis and secretion of stromal MMP-2 were increased in the presence of ovarian steroids. In contrast, expression of stromal MMP-9 was suppressed in the presence of ovarian steroids. No MMPs were synthesized in HEC-1A cells. Invasion assay revealed that HEC-1A cells achieved high tumoral invasiveness in the presence of beta-estradiol. CONCLUSIONS These findings suggest that stromal cell-derived MMP-2 is translocated to the surface of HEC-1A cells. Integrin beta 3 subunit might contribute, in part, to providing a binding site for MMP-2. Thus, HEC-1A cells invade by recruiting MMP-2 secreted by stromal cells, which is greatly enhanced in the presence of beta-estradiol. The 3D coculture provides an excellent experimental system in which single parameters can be isolated from a complex in vivo system in the process of endometrial cancer invasion.