Fibrinogen is a ligand for leukocyte integrin α_Mβ₂ (CD11b/CD18, Mac-1) and mediates adhesion and migration of leukocytes during the immune-inflammatory responses. The binding site for α_Mβ₂ resides in γC, a constituent subdomain in the D-domain of fibrinogen. The sequence γ383−395 (P2-C) in γC was implicated as the major binding site for α_Mβ₂. It is unknown why α_Mβ₂ on leukocytes can bind to immobilized fibrinogen in the presence of high concentrations of soluble fibrinogen in plasma. In this study, we have investigated the accessibility of the binding site in fibrinogen for α_Mβ₂. We found that the α_Mβ₂-binding site in γC is cryptic and identified the mechanism that regulates its unmasking. Proteolytic removal of the small COOH-terminal segment(s) of γC, γ397/405−411, converted the D₁₀₀ fragment of fibrinogen, which contains intact γC and is not able to inhibit adhesion of the α_Mβ₂-expressing cells, into the fragment D₉₈, which effectively inhibited cell adhesion. D₉₈, but not D₁₀₀, bound to the recombinant α_MI-domain, and the α_MI-domain recognition peptide, α_M(Glu²⁵³−Arg²⁶¹). Exposure of the P2-C sequence in fibrinogen, D₁₀₀, and D₉₈ was probed with a site-specific mAb. P2-C is not accessible in soluble fibrinogen and D₁₀₀ but becomes exposed in D₉₈. P2-C is also unmasked by immobilization of fibrinogen onto a plastic and by deposition of fibrinogen in the extracellular matrix. Thus, exposure of P2-C by immobilization and by proteolysis correlates with unmasking of the α_Mβ₂-binding site in the D-domain. These results demonstrate that conformational alterations regulate the α_Mβ₂-binding site in γC and suggest that processes relevant to tissue injury and inflammation are likely to be involved in the activation of the α_Mβ₂-binding site in fibrinogen.