Heparin catalysis of clotting proteinase inactivation occurs most efficiently through the reaction of the proteinase with the antithrombin-heparin complex. The efficiency of a heparin molecule in this reaction depends on the presence of a specific pentasaccharide sequence in it, and its molecular weight. The mechanism by which such high affinity heparin acts when antithrombin III is the inhibitor is promotion of the formation of an intermediate proteinase-heparin-antithrombin complex. Heparin promotion of thrombin inactivation by heparin cofactor II may occur by a similar mechanism. The requirement for a specific oligosaccharide sequence within the heparin molecule does not, however, exist for heparin cofactor II. Binding of heparin to both thrombin and antithrombin III interferes with thrombin inactivation. This binding is very dependent on the ionic strength of the reaction mixture and may explain some of the discordant results and interpretations from early studies on the mechanism of heparin action. Low ionic strength in in vitro reactions also results in cleavage of antithrombin III by thrombin in the presence of heparin and effectively converts antithrombin III from an inhibitor to a substrate.