A sulfamidase has been isolated from lymphoid tissues of rats, dogs, and man and purified by combined Sephadex gel filtration and gel electrophoresis. The enzyme which has an optimum pH of 5.0 is shown to catalyze the hydrolysis of a single N-linked sulfate residue from heparin and from hexa- and tetra-saccharides originating from the degradation of heparin by bacterial enzymes. The enzyme has no action upon N-sulfated disaccharides or glucosamine 2,6-disulfates or upon several sulfated mucopolysaccharides.The possible pathway for the complete desulfation of heparin, which occurs in vivo, is discussed on the basis of the properties of this sulfamidase.