We have fractionated crude porcine heparin to obtain highly active as well as relatively inactive species of molecular weight approximately 7000 with specific anticoagulant activities of 360 and 12 units/mg, respectively. Nitrous acid degradation of both of these polymers yielded a tetrasaccharide fraction, 1beta, that contained equimolar amounts of iduronic and glucuronic acids, possessed an internal N-acetylated glucosamine, and carried anhydromannitol at the reducing end position. The 1beta tetrasaccharide derived from the highly active heparin, 1betaa, was recovered in a yield of 1.1 mol/7000 daltons. Our analyses indicate that at least 95% of the 1betaa is a single structure that consists of the following unique monosaccharide sequence: L-iduronic acid --> N-acetylated D-glucosamine-6-sulfate --> D-glucuronic acid --> N-sulfate D-glucosamine-6-sulfate. The 1beta tetrasaccharide fraction from relatively inactive mucopolysaccharide, 1betai, was recovered in a yield of 0.3 mol/7000 daltons and was a mixture of several components. Only 8.5% of the 1betai tetrasaccharide fraction exhibited the same uronic acid placement and sulfate group position found in 1betaa. Thus, 2.6% of relatively inactive mucopolysaccharide molecules contain the unique tetrasaccharide sequence found within each molecule of highly active heparin. Given the correlation between abundance of this unique 1betaa tetrasaccharide sequence and biologic potency, we suggest that this structure represents the critical site responsible for anticoagulant activity.