Fibrinogen αC residues 242-424 have been shown to have a major regulatory role in the activation of factor XIII-A₂B₂ (FXIII-A₂B₂); however, the interactions underpinning this enhancing effect have not been determined. Here, we have characterized the binding of recombinant (r)FXIII-A subunit and FXIII-A₂B₂ with fibrin(ogen) and fibrin αC residues 233-425. Using recombinant truncations of the fibrin αC region 233-425 and surface plasmon resonance, we found that activated rFXIII-A bound αC 233-425 (K_d of 2.35 ± 0.09μM) which was further localized to αC 389-403. Site-directed mutagenesis of this region highlighted Glu396 as a key residue for binding of activated rFXIII-A. The interaction was specific for activated rFXIII-A and depended on the calcium-induced conformational change known to occur in rFXIII-A during activation. Furthermore, nonactivated FXIII-A₂B₂, thrombin-cleaved FXIII-A₂B₂, and activated FXIII-A₂B₂ each bound fibrin(ogen) and specifically αC region 371-425 with high affinity (K_d < 35nM and K_d < 31nM, respectively), showing for the first time the potential involvement of the αC region in binding to FXIII-A₂B₂. These results suggest that in addition to fibrinogen γ′ chain binding, the fibrin αC region also provides a platform for the binding of FXIII-A₂B₂ and FXIII-A subunit.