Different β1 integrins bind Arg. Gly-Asp (RGD) peptides with differing specificities, suggesting a role for residues in the α subunit in determining ligand specificity. Integrin α5β1 has been shown to bind with high affinity to peptides containing an Arg-Gly-Asp-Gly-Trp (RGDGW) sequence but with relatively low affinity to other RGD peptides. The residues within the ligand-binding pocket that determine this specificity are currently unknown. A cyclic peptide containing the RGDGW sequence was found to strongly perturb the binding of the anti-α5 monoclonal antibody (mAb) 16 to α5β1. In contrast, RGD peptides lacking the tryptophan residue acted as weak inhibitors of mAb 16 binding. The epitope of mAb 16 has previously been localized to a region of the α5 subunit that contains Ser156-Trp157. Mutation of Trp157 (but not of Ser156 or surrounding residues) to alanine blocked recognition of mAb 16 and perturbed the high affinity binding of RGDGW-containing peptides to α5β1. The same mutation also abrogated recognition of the α5β1-specific ligand peptide Arg-Arg-Glu-Thr-Ala-Trp- Ala (RRETAWA). Based on these findings, we propose that Trp157 of α5 participates in a hydrophobic interaction with the tryptophan residue in RGDGW, and that this interaction determines the specificity of α5β1 for RGDGW-containing peptides. Since the RGD sequence is recognized predominantly by amino acid residues on the β1 subunit, our results suggest that Trp157 of α5 must lie very close to these residues. Our findings therefore provide new insights into the structure of the ligand-binding pocket of α5β1.