Proteins with affinity to heparin under physiological conditions were isolated from bovine cerebral cortex. First, the extract of cerebral cortex was applied to a chondroitin polysulfate column under physiological conditions. Then, the pass-through fraction was applied to a heparin column. Among the bands on SDS polyacrylamide gel electrophoresis of the fraction bound to the heparin column, the major one was identified as fructose 1,6-bisphosphate aldolase (FPA), a cytosolic enzyme involved in the glycolytic pathway. The results indicated that FPA is a heparin-binding protein which exhibits no affinity to chondroitin polysulfate. The results of affinity chromatographies revealed that FPA binds to intact heparin and modified heparins desulfated at C2 OH of the iduronic acid residue or at C6 OH or C2 NH2 of the glucosamine residue. When 6-O-desulfated heparin was employed as the affinity ligand, a single peak having FPA activity was isolated from the extract of bovine cerebral cortex. By further Mono Q chromatography and Superdex gel-filtration, five isoenzymes were purified with more than 50% recovery. These isoenzymes were identified as FPA A4, A3C1, A2C2, A1C3, and C4 by native electrophoresis with and without 4 M urea and subsequent amino acid sequence analysis. The use of 6-O-desulfated heparin affinity chromatography thus facilitated the purification of FPA.