The interaction between the central cell-binding domain (CBD) of fibronectin (FN) and its receptor integrin α 5 β 1 was analyzed by both ligand-binding and cell adhesion assays. The ligands used were a CBD fragment (pCBD) of human plasma fibronectin and its recombinant versions including wild type CBD (wtCBD) and its two mutants (CBD-I, lacking the integrin recognition sequence Arg-Gly-Asp from wtCBD and CBD-II, missing the synergistic regions). The ligand-binding assay showed that CBD-I and CBD-II bind to the receptor, although the binding ability of the former was weaker than that of the latter. The affinity of pCBD to the receptor was much higher than the two mutants. The cell adhesion assay also revealed that cells were able to attach and spread on CBD-I to the same extent as on CBD-II, although the extent of spreading on the two mutant polypeptides was less than 4.1% of pCBD or wtCBD. On the other hand, β 1 -dependent cell spreading on CBD-II was not inhibited by the monoclonal antibody specific for the α 5 subunit, while that on CBD-I, wtCBD, or pCBD was inhibited by the same antibody. The present study suggests that the α 5 subunit does not participate in direct binding to the Arg-Gly-Asp site in CBD when cells adhere to FN through the integrin α 5 β 1 , but that it is involved in the interaction with the synergistic regions of CBD, which then enhances the binding of the β 1 subunit to the Arg-Gly-Asp sequence containing CBD.