Ovarian carcinoma multicellular spheroids are an in vitro model of micrometastasis whose adhesive abilities have not been elucidated. In this study, we identified adhesion molecules that mediate the formation of ovarian carcinoma spheroids and their subsequent adhesion to extracellular matrix proteins. The NIH:OVCAR5, but not the SKOV3, ovarian carcinoma cell line formed spheroids similar to multicellular aggregates isolated from patient ascitic fluid. NIH:OVCAR5 spheroid formation was augmented by a beta 1-integrin-stimulating monoclonal antibody or exogenous fibronectin, but was inhibited by blocking monoclonal antibodies against the alpha 5- or beta 1-integrin subunits. By immunohistochemical staining, alpha 2-, alpha 3-, alpha 5-, alpha 6-, and beta 1-integrin subunits, CD44, and fibronectin were detected in NIH:OVCAR5 spheroids. NIH:OVCAR5 spheroids adhered to fibronectin, laminin, and type IV collagen, and this adhesion was partially inhibited by blocking antibodies against the alpha 5-, alpha 6-, and alpha 2-integrin subunits, respectively. A blocking monoclonal antibody against the beta 1-integrin subunit completely inhibited adhesion of the spheroids to all three proteins. These results suggest that interactions between the alpha 5 beta 1-integrin and fibronectin mediate the formation of ovarian carcinoma spheroids and that their adhesion to extracellular matrix proteins at sites of secondary tumor growth may be mediated by a complex interaction between multiple integrins and their ligands.