Background: Lipopolysaccharide (LPS) is the primary pathogenic factor in Gram-negative sepsis. LPS is a polyanionic molecule that may act as a site of initiation of coagulation by activating factor (F)XII. While the physicochemical properties of LPS are known to be sensitive to interactions with divalent cations, it is unknown whether divalent cations modulate the activation of FXII by LPS. Aims: To determine whether divalent cations regulate the kinetics of FXII activation by select LPS chemotypes from E. coli . Methods: Aggregates of E. Coli LPS chemotypes O26:B6 and Rd2 were prepared in ultrapure water in the presence or absence of calcium (Ca 2+ ). The biophysical characterization of LPS chemotypes were determined by dynamic light scattering (DLS) and measurement of zeta potential (ZP). Chromogenic substrate assays were used to study the effects of LPS on FXII activation. Results: The LPS O26:B6 and Rd2 in the absence of Ca 2+ produced aggregates in form of micelles with hydrodynamic diameters of 190±10 and 200±7 nm and ZP of -25±0.8 and -60±2 mV, respectively. The presence of Ca 2+ produced aggregates in the form of vesicles with hydrodynamic diameters of 400±15 and 1300±60 nm and ZP of –11±2 and –3±0.5 mV, respectively. FXII was incubated in HEPES buffer with the distinct LPS morphologies in varying salt concentrations. We found maximal autoactivation of FXII by O26:B6 and Rd2 chemotypes at lower NaCl concentrations. The presence of Ca 2+ , however, selectively reduced the ability of O26:B6 to activate FXII. Strikingly, the neutralization of the surface net charge of the LPS chemotype Rd2 by Ca 2+ ions abrogated the amidolytic activity of Rd2. Conclusions: The presence of calcium ions induces changes in the physicochemical properties of LPS that result in distinct effects on FXII activation in vitro . We are currently studying whether this phenomena is conserved for other divalent cations including Zn, Mg, Cu, and Mn.