Fibronectin (FN)-mediated cell adhesion is controlled mainly by alpha 5 beta 1 (recognizing the RGD sequence) and alpha 4 beta 1 (recognizing the CS-1 peptide sequence of FN) integrin receptors. Integrin-dependent cell adhesion to FN is greatly promoted by optimal GM3 concentration at the surface membrane (Zheng, M., Fang, H., Tsuruoka, T., Tsuji, T., Sasaki, T., and Hakomori, S. (1993) J. Biol. Chem. 268, 2217-2222), and cell adhesion mediated by alpha 4 beta 1 (to FN) or alpha 6 beta 1 (to laminin) is inhibited by modifying N-glycosylation processing of the integrin receptor (e.g. Akiyama, S. K., Yamada, S. S., and Yamada, K. M. (1989) J. Biol. Chem. 264, 18011-18018). We therefore studied the specific role of N-glycosylation in alpha 5 beta 1 function. Key findings of the present study were as follows. (i) Adhesion of K562 cells to FN-coated plates, which is mediated solely by alpha 5 beta 1, was inhibited when cells were treated with a mixture of endo-N-acetylglucosaminidase F and peptide -N4-(N-acetylglucosaminyl)asparagine amidase F (endo-F/PNGase-F). (ii) The alpha 5 beta 1 receptor at the K562 cell surface tended to dissociate into alpha 5 and beta 1 subunits when an extract of cells treated with endo-F/PNGase-F was precipitated by integrin subunit-specific antibodies, i.e. the alpha 5 subunit was preferentially precipitated by anti-alpha 5 monoclonal antibody ZH5, and the beta 1 subunit was preferentially precipitated by anti-beta 1 monoclonal antibody ZH1. When intact cells were extracted and treated with either ZH5 or ZH1, both alpha 5 and beta 1 were coprecipitated, indicating that the two subunits are normally tightly associated with each other. (iii) Adhesion of alpha 5 beta 1-containing liposomes (phosphatidylcholine:cholesterol liposomes incorporating purified alpha 5 beta 1) to FN-coated plates was abolished by treatment of liposomes with endo-F/PNGase-F. Liposomes incorporating alpha 5 beta 1 pretreated with endo-F/PNGase-F also did not bind to FN. When purified alpha 5 beta 1 receptor was treated with endo-F/PNGase-F followed by ZH5 or ZH1, the alpha 5 or beta 1 subunit was precipitated separately, respectively. In contrast, both subunits were always coprecipitated when intact purified alpha 5 beta 1 receptor was directly treated with ZH5 or ZH1. These findings indicate that N-glycosylation of both the alpha and beta subunits of the alpha 5 beta 1 integrin receptor is essential for association of these subunits and for optimal binding to FN.