N-Acetylglucosamine-6-sulfate sulfatase, which liberates sulfate from the N-acetylglucosamine 6-sulfate residue at the nonreducing terminus of a 3H-labeled trisaccharide prepared from heparan sulfate, was purified 136-fold from human urine. The final N-acetylglucosamine-6-sulfate sulfatase preparation was free of all lysosomal sulfatases known to act on sulfated polysaccharides and gave a single band in polyacrylamide gel electrophoresis. The enzyme appears to be a glycoprotein with a molecular weight of around 97,000 and displays considerable charge heterogeneity. Multiple forms with pI values between 5.4 and 8.3 with a maximum at pH 7.7 were detected. The enzyme acts on the 3H-trisaccharide with a pH optimum at 5.5 and is active towards the sulfated monosaccharides N-acetylglucosamine 6-sulfate and glucose 6-sulfate. Although predominantly in exosulfatase, the enzyme catalyzes hydrolysis of sulfate from internal N-acetylglucosamine 6-sulfate moieties at a low rate. The Km for the 3H-trisaccharide, N-acetylglucosamine 6-sulfate, and glucose 6-sulfate were 0.15, 1.5, and 7.7 mM, respectively. The enzyme is inhibited by albumin, Hg2+, PO43-, SO42-, and CN-. Enzyme activity was highest in kidney and cultured fibroblasts but could be demonstrated in all human tissues tested.