The interaction of human plasma fibrinogen with leukocyte integrins α_Mβ₂ (CD11b/CD18, Mac-1) and α_Xβ₂ (CD11c/CD18, p150,95) is an important component of the inflammatory response. Previously, it was demonstrated that binding of fibrinogen to these integrins is mediated by γC, the globular C-terminal domain of the γ chain. In this study, evidence was found of another fibrinogen domain that can serve as a ligand for the 2 leukocyte integrins: α_EC, a homologous domain that extends the α chains in a recently discovered subclass of fibrinogen known as fibrinogen-420. Recombinant α_EC supported strong adhesion and migration of cells expressing α_Mβ₂ and α_Xβ₂, including nonactivated and activated U937 and THP-1 monocytoid cells, and neutrophils. Cells transfected with complementary DNA for these integrins also bound α_EC. The specificity of interaction was substantiated by inhibition of cell adhesion with antibodies against α_M, α_X, and β₂subunits. Also, neutrophil inhibitory factor, a specific inhibitor of α_Mβ₂ and α_Xβ₂function, efficiently blocked cell adhesion to α_EC. In α_Mβ₂ and α_Xβ₂, the I domain is the binding site for α_EC, since α_EC bound to recombinant α_M I and α_XI domains in a dose-dependent and saturable manner. Synthetic peptides that duplicated sequences γ190 to 202 and γ377 to 395, previously considered putative binding sites in γC, effectively inhibited α_Mβ₂- and α_Xβ₂-mediated adhesion to α_EC, suggesting that recognition of α_EC by the I domain involves structural features in common with those of γC. These findings identify α_EC as a second domain in fibrinogen-420 that binds α_Mβ₂ and α_Xβ₂ and can mediate leukocyte adhesion and migration.