The omentum is a major site of ovarian cancer metastasis. Our goal was to establish a three-dimensional (3D) model of the key components of the omental microenvironment (mesothelial cells, fibroblasts and extracellular matrices) to study ovarian cancer cell adhesion and invasion. The 3D model comprised of primary human fibroblasts extracted from normal human omentum, mixed with ECM and covered by a layer of primary human mesothelial cells, also from normal human omentum. After addition of ovarian cancer cells, the histological appearance of the 3D culture mimicked microscopic metastases to the omentum from patients with ovarian cancer. When ovarian cancer cells, SKOV3ip.1 and HeyA8, were applied to the 3D omental culture, 60% and 68% of all cells attached, respectively, but only 18% and 25% were able to invade. Ovarian cancer cells preferentially adhered to and invaded collagen I, followed by binding to collagen IV, fibronectin, vitronectin, laminin 10 and 1. Omental mesothelial cells significantly inhibited ovarian cancer cell adhesion and invasion, while omental fibroblasts induced adhesion and invasion. This effect is clearly mediated by direct cell-cell contact, since conditioned medium from mesothelial cells or fibroblasts has a minimal, or no, effect on ovarian cancer cell adhesion and invasion. In summary, we have established a 3D model to study the early steps of ovarian cancer metastasis to the human omentum, and found that omental mesothelial cells inhibit, while omental fibroblasts and the ECM enhance, the attachment and invasion of ovarian cancer cells. ' 2007 Wiley-Liss, Inc.Key words: 3-dimensional culture; 3D; tumor-stroma interaction; metastasis; ovarian cancer Ovarian cancer is typically diagnosed at an advanced stage, because most patients develop clinical symptoms late in the course of the disease, after the tumor has metastasized.1 The most common sites of metastasis from the primary ovarian tumor are the abdominal peritoneum and the omentum. Often the pliable, semitransparent omental tissue is transformed into a dense omental ''cake'' that impinges on the stomach and the transverse colon, causing a bowel obstruction. Even after infragastric removal of the omentum, which is regularly performed for ovarian cancer staging or debulking at the time of surgery, recurrent tumor cells home to residual areas of the omentum at the splenic hilum, the stomach and the transverse colon causing the reappearance of significant clinical symptoms.The majority of ovarian cancers are of epithelial origin, arising from the single layer of cells that covers the ovary, which is separated by a basement membrane from the underlying ovarian stroma.2,3 There are features unique to ovarian cancer that might facilitate its intraperitonel (ip) spread. The ovarian epithelium can adopt a mesenchymal phenotype characterized by cells that lack tight junctions, which makes them prone to exfoliation. 4 Once an ovarian epithelial cell undergoes transformation it is free to disseminate throughout the peritoneal cavity, carried b...