N-Acetylglucosamine-6-sulfate sulfatase (NG6SS) is an enzyme that catalyzes the hydrolysis of sulfate esters from the C-6 hydroxyl of N-acetylglucosamine. We report our purification and characterization of the enzyme and the discovery that it can remove sulfate from internally sulfated GlcNAc on glycopeptides and glycoproteins. The enzyme was purified from bovine kidney over 200,000-fold using a combination of ion-exchange and size-exclusion chromatography. NG6SS is soluble and occurs as a single subunit with apparent solution molecular weight of 60.2 kDa on gel filtration chromatography and approximately 52.5 and 57.8 kDa on reducing and nonreducing SDS/PAGE, respectively. The enzyme is highly basic and exhibits a broad pH range with an optimum at pH 6.5 and a temperature optimum of 37 degrees C. Among the mono- and disaccharide sulfates tested, only GlcNAc-6-SO4 is an effective substrate with a Km of 4.7 mM, and either free sulfate or phosphate inhibits the activity. Unexpectedly, we found that the enzyme displays endosulfatase activity and quantitatively releases 35SO4 from 35SO4-labeled glycopeptides and intact glycoproteins isolated from human Molt-3 cells, which we have previously shown to synthesize glycoproteins containing GlcNAc-6-SO4 residues within the sequence Gal beta 1-4[SO-3-6]-GlcNAc beta 1-R of complex-type N-linked oligosaccharides. The N-terminal sequence of the bovine NG6SS was homologous to a human-liver-derived N-acetylglucosamine-6-sulfatase. The endosulfatase activity of bovine kidney NG6SS may be important in its potential role in the degradation of sulfated glycans and may make this enzyme a valuable reagent to study the biological functions of sulfated glycoproteins.