Human heparanase is an endo--D-glucuronidase that degrades heparan sulfate/heparin and has been implicated in a variety of biological processes, such as inflammation, tumor angiogenesis, and metastasis. Although the cloned enzyme has been demonstrated to have a critical role in tumor metastasis, the substrate specificity has been poorly understood. In the present study, the specificity of the purified recombinant human heparanase was investigated for the first time using a series of structurally defined oligosaccharides isolated from heparin/heparan sulfate. The best substrates were GlcN(NS,6S) (where HexUA represents hexuronic acid) has been proposed as a probable physiological target octasaccharide sequence. These findings will aid establishing a quantitative assay method using the above tetrasaccharide and designing heparan sulfate-based specific inhibitors of the heparanase for new therapeutic strategies.