A homogeneous set of low-molecular weight heparins, chemically modified to yield different degrees of sulfation, were investigated for their ability to interfere with the antithrombin (AT)-factor Xa (FXa) interaction process in the presence or absence of physiological concentrations of calcium ions. The heparin-AT dissociation constants were not appreciably affected by the presence of the metal ion, whereas the catalytic process was strongly dependent on Ca 2+. Our data suggest that AT binding to heparin represents the main factor driving the FXa inhibition process. In addition, the presence of the metal ion is likely to mask favorable AT- heparin ionic contacts occurring with the highly sulfated material. These results help in assessing proper structure-activity relationships for glycosaminoglycans, a multitarget family of biologically active compounds.