A two-dimensional immunoelectrophoretic method has been used to obtain information on the binding of heparin to purified antithrombin III and the antithrombin III--thrombin complex. The difference in mobility of the components in a gel containing heparin enables distinction between free and complexed forms of antithrombin III. The results obtained with purified preparations show that heparin is bound more strongly to antithrombin III than to the antithrombin III--thrombin complex. In plasma heparin is bound to several components, only a fraction being bound to antithrombin III. Several components containing antithrombin III are detectable in serum.