A new, highly discriminating affinity chromatographic technique has been developed which employs antithrombin and concanavalin A-Sepharose to fractionate heparin species of all molecular sizes. This methodology is able to subdivide the active mucopolysaccharide pools of molecular weight 6,000 to 8,000 (LMW) or 18,000 to 22,000 (HMW) into various species with descending affinities for antithrombin as well as decreasing anticoagulant potencies. The upper 10% of these two pools, either LMW or HMW highly active heparin, appears to be relatively homogeneous with respect to interactions with antithrombin and possessed anticoagulant potencies of 350 units/mg and 731 units/mg, respectively. The HMW highly active heparin has been examined by analytic ultracentrifugation. It exhibited a charge-connected weight-average molecular weight of 22,000 +/- 2,000 with minimal size heterogeneity. The stoichiometries of interaction of antithrombin and platelet factor 4 with HMW highly active heparin as determined by fluorescence spectroscopy indicated that 2 molecules of either protein are able to bind to 1 molecule of the mucopolysaccharide. These studies also reveal that the binding of antithrombin to HMW highly active heparin is characterized by KDISSHAT = 5.0 X 10(-8) M and KDISSHAT2 = 1.0 x 10(-7) M, respectively. The avidity of platelet factor 4 for HMW highly active heparin could not be quantitated but appears to be at least 10 to 100 times greater than that of antithrombin for mucopolysaccharide.