Heparan sulfate glycosaminoglycans are structurally complex polysaccharides critically engaged in a wide range of cell and tissue functions. Any structure-based approach to study their respective biological functions is facilitated by the use of select heparan sulfate glycosaminoglycan-degrading enzymes with unique substrate specificities. We recently reported of one such enzyme, the ⌬4,5-glycuronidase cloned from Flavobacterium heparinum and recombinantly expressed in Escherichia coli (Myette, J. R., Shriver, Z., Kiziltepe, T., McLean, M. W., Venkataraman, G., and Sasisekharan, R. (2002) Biochemistry 41, 7424 -7434). In this study, we likewise report the molecular cloning of the 2-O-sulfatase from the same bacterium and its recombinant expression as a soluble, highly active enzyme. At the protein level, the flavobacterial 2-O-sulfatase possesses considerable sequence homology to other members of a large sulfatase family, especially within its amino terminus, where the highly conserved sulfatase domain is located. Within this domain, we have identified by sequence homology the critical active site cysteine predicted to be chemically modified as a formylglycine in vivo. We also present a characterization of the biochemical properties of the enzyme as it relates to optimal in vitro reaction conditions and a kinetic description of its substrate specificity. In particular, we demonstrate that in addition to the fact that the enzyme exclusively hydrolyzes the sulfate at the 2-O-position of the uronic acid, it also exhibits a kinetic preference for highly sulfated glucosamines within each disaccharide unit, especially those possessing a 6-O-sulfate. The sulfatase also displays a clear kinetic preference for disaccharides with ␤134 linkages but is able, nevertheless, to hydrolyze unsaturated, 2-O-sulfated chondroitin disaccharides. Finally, we describe the substrate-product relationship of the 2-O-sulfatase to the ⌬4,5-glycuronidase and the analytical value of using both of these enzymes in tandem for elucidating heparin/heparan sulfate composition.