Epithelial cancer of the ovary spreads by implantation of tumor cells onto the mesothelial lining of the peritoneal cavity. We have developed an in vitro binding assay using confluent monolayers of normal peritoneal mesothelial cells in order to assess the role of known adhesion proteins in this process. Cells from normal ovarian surface epithelium and the ovarian cancer cell lines CAOV-3 and SKOV-3 exhibited significant adhesion to mesothelium in vitro (range 33–56% specific binding). Although these cells expressed several adhesion molecules, including CD44 and integrins such as α4β1, α5β1, and ανβ3, only anti-CD44 antibody was capable of inhibiting mesothelial binding (range 42–44% inhibition). Adhesion molecule expression was also determined for fresh ovarian specimens, with CD44 being expressed in 2 of 2 cases of normal ovarian epithelium, 15 of 16 (94%) cases of tissue-derived tumor (from primary sites or peritoneal implants), and only 2 of 8 (25%) cases of free-floating tumor cells from ascites. Three of three CD44-positive cases derived from peritoneal implants exhibited significant mesothelial binding which was partly blocked by anti-CD44 antibody, whereas 2 of 2 CD44-negative cases derived from ascites showed minimal binding. CD44-mediated binding of ovarian cancer cells was determined to be due to recognition of mesothelium-associated hyaluronate, suggesting that the CD44H isoform was involved in this process. Immunoprecipitation of the CD44 species expressed by ovarian cancer cells revealed 2 major bands at 85–90 and 180 kDa, consistent with the known molecular masses of CD44H. These results suggest that CD44H may be an important mediator of ovarian cancer cell implantation and that decreased CD44H expression may be associated with release of cells into the peritoneal space during ascites formation. It is possible that strategies to interfere with CD44H function may result in decreased intraabdominal spread of this highly lethal neoplasm.