Fibrinogen interactions with vascular endothelial cells are implicated in various physiological and pathophysiological events, including angiogenesis and wound healing. We have shown previously that integrin α₅β₁ is a fibrinogen receptor on endothelial cells [Suehiro, K, Gailit, J., and Plow, E. F. (1997) J. Biol. Chem, 272, 5360-5366]. In the present study, we have characterized fibrinogen interactions with purified α₅β₁ and have identified the recognition sequence in fibrinogen for α₅β₁. The binding of fibrinogen to immobilized α₅β₁ was selectively supported by Mn²⁺. Fibrinogen bound to purified α₅β₁ in a time-dependent, specific, and saturable manner in the presence of Mn²⁺, and the binding was blocked completely by Arg-Gly-Asp (RGD)-containing peptides and by anti-α₅ and anti-α₅β₁ monoclonal antibodies. A monoclonal antibody directed to the C-terminal RGD sequence at Aα572-574 significantly inhibited the binding of fibrinogen to α₅β₁, whereas monoclonal antibodies directed to either the N-terminal RGD sequence at Aα95-97 or the C-terminus of the γ-chain did not. Furthermore, substituting RGE for RGD at position Aα95-97 in recombinant fibrinogen had a minimal effect on binding, whereas substituting RGE for RGD at position Aα572-574 decreased binding by 90%. These results demonstrate that the C-terminal RGD sequence at Aα572-574 is required for the interaction of fibrinogen with α₅β₁.