Heparinase (heparin lyase, EC 4.2.2.7) prepared from Flavobacterium heparinum was used to digest heparin. The products of digestion were examined with a viscosometric assay at various stages of the reaction to measure their average molecular weight. By comparison with computer simulations of various models, heparinase was shown to act in a random endolytic mode. The relative abundance of intermediates in heparin degradation catalyzed by heparinase immobilized on Sepharose 4B was measured by high pressure liquid chromatography (HPLC) at various time points. The results obtained using HPLC were consistent with a random endolytic mechanism. The heparin digestion products were separated and identified using gel permeation chromatography. The final distributions of heparin degradation products for free and immobilized heparinase were identical. Contaminating sulfatases and glycuronidases which could have subsequently acted on heparin degradation products were not found in significant amounts in the heparinase preparation studied.