T o better understand how heparin structure affects its activity the relationships between the functional domains for inhibitor binding and charge density were investigated to determine how these domains affect heparin-mediated thrombin inhibition by two different heparin-dependent protease inhibitors, antithrombin (AT) and heparin cofactor II (HC II). A series of heparins, fractionated systematically by charge density, was further fractionated on antithrombin agarose to isolate more homogeneous subfractions that were either inactive or highly active with respect to thrombin inhibition by AT. With AT, the activities of the AT-active subfractions increased sharply with heparin charge density, while those with little or no affinity for AT were virtually inactive. In contrast, with HC II inhibitor, the activities of the heparins depended only upon their charge densities and were independent of AT affinity. At any given charge density, the heparin before fractionation by AT affinity and the fractions that were highly active and inactive with AT were all equally active with HC II. The two inhibitors also differed in their reactivity with heparan sulfate and dermatan sulfate. A charge-density effect with the subfractions having similar high affinity for AT demonstrates that charge density represents a heparin functional domain that is independent of the AT-binding domain. The behavior of the AT-inactive heparins, being fully active with HC II, demonstrates the functional domain necessary for AT binding is not needed to produce HC II activity.