Fibronectin (Fn) binding to the integrins alpha IIb beta 3 and alpha v beta 3 involves the Arg-Gly-Asp sequence. The identification of other regions of Fn that interact with alpha IIb beta 3 suggests a potential mechanism for differential ligand recognition by integrins. We report here the identification of an 11-residue peptide sequence from the 9th type III repeat of Fn (3Fn9), which inhibits ligand binding to alpha IIb beta 3 by interacting directly with this receptor. Mutational analysis demonstrated that this same region was involved in the formation of epitopes for two anti-Fn mAbs that inhibit Fn binding to alpha IIb beta 3, thus emphasizing the role of this site in the macromolecule. Molecular modeling of the 3Fn9-10 modules suggested that Fn residues Asp1373-Thr1383 are at least 25 A distant from the Arg-Gly-Asp site and therefore does not directly interact with it. The 3Fn9 site was differentially recognized by the beta 3 integrin family. The Asp1373-Thr1383 peptide failed to inhibit ligand binding to alpha v beta 3, a recombinant Fn Ala1235-Ser1436 fragment was not recognized by alpha v beta 3, and addition of the 3Fn9 module to the amino terminus of the 3Fn10 did not affect the potency of inhibition of Fn binding to alpha v beta 3. Thus, a novel integrin recognition site in the 3Fn9 module of Fn that is differentially recognized by the beta 3 integrins has been localized within the residues Asp1373-Thr1383.