Coagulation factor XIII (FXIII) stabilizes fibrin fibers and is therefore a major player in the maintenance of hemostasis. FXIII is activated by thrombin resulting in cleav- age and release of the FXIII activation peptide (AP-FXIII). The objective of this study was to characterize the released AP-FXIII and determine specific features that may be used for its specific detec- tion. We analyzed the structure of bound AP-FXIII within the FXIII A-subunit and interactions of AP-FXIII by hydrogen bonds with both FXIII A-subunit mono- mers. We optimized our previously devel- oped AP-FXIII ELISA by using 2 monoclo- nal antibodies. We determined high binding affinities between the antibodies and free AP-FXIII and demonstrated spe- cific binding by epitope mapping analy- ses with surface plasmon resonance and enzyme-linked immunosorbent assay. Be- cause the structure of free AP-FXIII had been characterized so far by molecular modeling only, we performed structural analysis by nuclear magnetic resonance. Recombinant AP-FXIII was largely flexible both in plasma and water, differing signifi- cantly from the rigid structure in the bound state. We suggest that the recog- nized epitope is either occluded in the noncleaved form or possesses a struc- ture that does not allow binding to the antibodies. On the basis of our findings, we propose AP-FXIII as a possible new marker for acute thrombotic events.