Factor XIII catalyzes formation of γ-glutamyl-ε-lysyl crosslinks within fibrin clots. FXIII A 2 can be activated proteolytically with thrombin and low mM Ca 2+ or nonproteolytically with high monovalent/divalent cations along with low mM Ca 2+ . Physiologically, FXIII A 2 is poised to respond to transient influxes of Ca 2+ in a Na + containing environment. A successful strategy to monitor FXIII conformational events is hydrogen–deuterium exchange (HDX) coupled with mass spectrometry. FXIII A 2 was examined in the presence of different cations (Ca 2+ , Mg 2+ , Ba 2+ , Cu 2+ , Na + , TMAC + , and EDA 2+ ) ranging from 1 to 2mM, physiological Ca 2+ concentration, to 50–500mM for nonproteolytic activation. Increases in FXIII solvent exposure could already be observed at 1mM Ca 2+ for the dimer interface, the catalytic site, and glutamine substrate regions. By contrast, solvent protection was observed at the secondary cleavage site. These events occurred even though 1mM Ca 2+ is insufficient for FXIII activation. The metals 1mM Mg 2+ , 1mM Ba 2+ , and 1mM Cu 2+ each led to conformational changes, many in the same FXIII regions as Ca 2+ . FXIII could also be activated nonproteolytically with 500mM tetramethylammonium chloride (TMAC + ) and 500mM ethylenediamine (EDA 2+ ), both with 2mM Ca 2+ . These different HDX studies help reveal the first FXIII segments that respond to physiological Ca 2+ levels.