The interaction between the leukocyte integrin α_Mβ₂ (CD11b/CD18, Mac-1, CR3) and fibrinogen mediates the recruitment of phagocytes during the inflammatory response. Previous studies demonstrated that peptides P2 and P1, duplicating γ377−395 and γ190−202 sequences in the γC domain of fibrinogen, respectively, blocked the fibrinogen-binding function of α_Mβ₂, implicating these sequences as possible binding sites for α_Mβ₂. To determine the role of these sequences in integrin binding, recombinant wild-type and mutant γC domains were prepared, and their interactions with the α_MI-domain, a ligand recognition domain within α_Mβ₂, were tested. Deletion of γ383−411 (P2-C) and γ377−411 produced γC mutants which were defective in binding to the α_MI-domain. In contrast, alanine mutations of several residues in P1 did not affect α_MI-domain binding, and simultaneous mutations in P1 and deletion of P2 did not decrease the binding function of γC further. Verifying the significance of P2, inserting P2-C and the entire P2 into the homologous position of the βC-domain of fibrinogen imparted the higher α_MI-domain binding ability to the chimeric proteins. To further define the molecular requirements for the P2-C activity, synthetic peptides derived from P2-C and a peptide array covering P2-C have been analyzed, and a minimal recognition motif was localized to γ³⁹⁰NRLTIG³⁹⁵. Confirming a critical role of this sequence, the cyclic peptide NRLTIG retained full activity inherent to P2-C, with Arg and Leu being important residues. Thus, these data demonstrate the essential role of the P2, but not P1, sequence for binding of γC by the α_MI-domain and suggest that the adhesive function of P2 depends on the minimal recognition motif NRLTIG.