Seven pharmaceutical heparins were investigated by oligosaccharide mapping by digestion with heparin lyase 1, 2, or 3, followed by high performance liquid chromatography analysis. The structure of one of the prepared mapping standards, ΔUA-Gal-Gal-Xyl-O-CH(2)CONHCH(2)COOH (where ΔUA is 4-deoxy-α-l-threo-hex-4-eno-pyranosyluronic acid, Gal is β-d-galactpyranose, and Xyl is β-d-xylopyranose) released from the linkage region using either heparin lyase 2 or heparin lyase 3 digestion, is reported for the first time. A size-dependent susceptibility of site cleaved by heparin lyase 3 was also observed. Heparin lyase 3 acts on the undersulfated domains of the heparin chain and does not cleave the linkages within heparin's antithrombin III binding site. Thus, a novel low molecular weight heparin (LMWH) is afforded on heparin lyase 3 digestion of heparin due to this unique substrate specificity, which has anticoagulant activity comparable to that of currently available LMWH.