2-Deoxy-D-glucose (dGlc) is a carbohydrate with significant activity as an inhibitor of glucose metabolism and as a precursor in the synthesis of glycosylated macromolecules; several of the enzymes associated with its metabolism remain uncharacterized. In the present report, the partial purification and some of the properties of a mammalian enzyme that appears to be relatively specific for the hydrolysis of dGlc bound in glycosidic linkage is described. The physiological function of this enzymatic activity is unknown. In addition, dGlc has been shown to be taken up by HTC cells in culture and incorporated into macromolecular bound form, both as dGlc and as 2-deoxygalactose which is formed from dGlc.