The γ chain of human plasma fibrinogen is heterogeneous with three forms differing in length at the C-terminus. Alternative RNA splicing produces two γ chain mRNAs encoding γ50 and γ57.5 polypeptides, while fibrinogen γ55 is produced by post-translational modification of the γ57.5 chain. The composition of purified variant γ chain fibrinogens, which comprise 10% to 13% total plasma fibrinogen, is predominantly heterodimeric (Aα, Bβ, γ50/γ55 or Aα, Bβ, γ50/γ57.5), whereas the composition of purified fibrinogen with the major form of the γ chain is homodimeric (Aα, Bβ, γ50/γ50). These γ chain variations interrupt sequences that mediate platelet-fibrinogen interactions. Therefore, the structure and function of γ57.5 C-terminal sequences were investigated using synthetic peptides and a specific monoclonal antibody (MoAb), L2B. The L2B epitope was localized and included γ57.5 chain residues 409-412 (Arg-Pro-Glu-His), as determined by differential enzyme-linked immunosorbent assay (ELISA) reactivity with a His-412 deleted synthetic peptide and by Western blot analysis of plasmin cleaved fibrinogen γ57.5. L2B had no effect on adenosine diphosphate (ADP)-induced platelet aggregation supported by either fibrinogen γ50 or γ57.5. High concentrations (0.5 to 1 mmol/L) of synthetic peptide γ57.5408-416 only weakly inhibited ADP-induced platelet aggregation supported by either fibrinogen γ50 or γ57.5. Binding of fibrinogen γ50 (IC50 = 780 μmol/L) or γ57.5 (IC50 = 650 μmol/L) to ADP-stimulated platelets was weakly inhibited, and MoAb L2B failed to inhibit fibrinogen γ57.5 binding. Peptide γ57.5408-416 failed to dissociate platelet-bound fibrinogens. These data indicate that the γ408-416 sequence of fibrinogen γ55 or γ57.5 alone is unlikely to bind to the platelet fibrinogen receptor, glycoprotein IIb-IIIa (GPIIb-IIIa), in support of platelet aggregation under physiologic conditions. The sequence recognized by L2B does not resemble known GPIIb-IIIa binding site peptide sequences [Arg-Gly-Asp-Ser (RGDS) or γ50400-411] as determined by competitive inhibition ELISA comparing these binding site synthetic peptides with γ57.5408-416. This epitope is available for binding MoAb L2B in γ55 or γ57.5 chain dimers and binds to all γ57.5408-416 epitopes equally in non-crosslinked and factor XIIIa crosslinked fibrin clots. The availability of the L2B epitope in crosslinked fibrin indicates that it is exposed in the fibrin clot, which is in agreement with the predicted secondary structure of the γ57.5 chain C-terminus.