Integrin adhesion receptors participate in two-way transfer of information across the plasma membrane. For example, cytoplasmic events, such as activation of protein kinase C, cause an increase in the fibrinogen (Fg) binding affinity of the extracellular domain of integrin alpha IIb beta 3 ("inside-out signaling"). Conversely, ligand binding to alpha IIb beta 3 results in the generation of intracellular signals. We used anti-LIBS2, an anti-beta 3 monoclonal antibody, to understand potential mechanisms of this bidirectional signaling. Anti-LIBS2 bound to alpha IIb beta 3 with low affinity (Kd = 7.4 microM), and mimicked inside-out signaling by promoting Fg binding. The affinity of anti-LIBS2 binding was increased 20-fold (Kd = 326 nM) by addition of an Fg-mimetic synthetic peptide, RGDS. Thus, anti-LIBS2 and ligands (Fg and Fg-mimetic peptides) bind cooperatively to integrin alpha IIb beta 3, indicating a functional linkage between the ligand-binding site and the antibody-binding site. The anti-LIBS2-binding site was mapped by its binding to proteolytic and recombinant fragments of the beta 3 subunit. The epitope was located within an 89-residue region immediately adjacent to the transmembrane domain and 400 residues carboxyl-terminal to the known ligand-binding site(s). Electron microscope images of rotary shadowed ternary complexes of Fg, anti-LIBS2, and alpha IIb beta 3 revealed that the ligand-binding site and anti-LIBS2 epitope are separated by about 16 nm. This indicates that propagated long distance conformational changes can occur in alpha IIb beta 3. Such changes are likely to be involved in the bidirectional signaling function of this integral membrane protein.