Ovarian cancer cells frequently metastasize by implanting onto the peritoneal mesothelial surface of the abdominal cavity. Although the CD44 molecule expressed by ovarian cancer cells is known to partly mediate this process, the role of other adhesion proteins such as β1-integrins has been previously difficult to demonstrate using the 4B4 anti-β1 neutralizing antibody. In view of the widespread expression of β1-integrins in ovarian cancer, however, we have further examined the potential role of this class of molecules in ovarian cancer cell implantation through the use of an alternative anti-β1 neutralizing antibody, MAB13. We now report that MAB13 is capable of inhibiting the binding of three separate human ovarian cancer cell lines (36M2, CAOV-3, SKOV-3) to mesothelium (mean 37 ± 4% inhibition, n = 21, P < 0.001). An additive inhibitory effect was observed when MAB13 was combined with anti-CD44 antibody (clone 515) (mean 63 ± 3% inhibition, n = 19, P < 0.001), suggesting that binding occurs through two independent pathways involving both β1-integrins and CD44. Because fibronectin is an extracellular matrix ligand recognized by many types of integrins and is abundantly expressed on mesothelial cells, the inhibitory effects of MAB13 and 4B4 on ovarian cancer cell binding to fibronectin were directly compared. MAB13 inhibited ovarian cancer cell binding to fibronectin to a significantly greater degree than did 4B4, suggesting that the discordant effects of these two antibodies on mesothelial adhesion may be partly related to their differential ability to neutralizing fibronectin-mediated binding. Studies using anti-α5 neutralizing antibody demonstrated that the α5β1 fibronectin receptor contributes to approximately 50% of integrin-mediated binding of 36M2 and CAOV-3 cells (which express the α5 chain in 54 and 58% of cells, respectively). Since the RGD sequence of fibronectin is a known recognition site for many types of integrins, including α5β1 and ανβ3, we performed binding in the continued presence of both anti-α5 and RGD peptide in order to exclude other types of fibronectin–integrin interactions. The addition of RGD peptides at concentrations known to be capable of blocking fibronectin binding resulted in no additional inhibitory effect over that observed with anti-α5 antibody alone, suggesting that α5β1 was the major receptor responsible for fibronectin-mediated ovarian cancer binding to mesothelium. These data demonstrate that ovarian cancer cell binding to peritoneal mesothelium is mediated by several adhesion pathways and that simultaneous inhibition of both β1-integrin and CD44 function may be necessary in order to significantly limit the intraabdominal spread of this tumor in vivo.