TheO-GlcNAcmodificationinvolvestheattachmentofsingle-O-linkedN-acetylglucosamine residues to serine and threo-nine residues of nucleocytoplasmic proteins. Interestingly, pre-vious biochemical and structural studies have shown thatO-GlcNAcase (OGA), the enzyme that removesO-GlcNAc fromproteins, has an active site pocket that tolerates variousN-acylgroups in addition to theN-acetyl group of GlcNAc. Theremarkable sequence and structural conservation of residuescomprising this pocket suggest functional importance. Wehypothesized this pocket enables processing of metabolic vari-ants ofO-GlcNAc that could be formed due to inaccuracy withinthe metabolic machinery of the hexosamine biosynthetic path-way. In the accompanying paper (Bergfeld, A. K., Pearce, O. M.,Diaz, S. L.,Pham, T., and Varki, A. (2012)J. Biol. Chem.287,28865–28881),N-glycolylglucosamine (GlcNGc) wasshown to be acatabolite of NeuNGc. Here, we show that the hexosamine sal-vage pathway can convert GlcNGc to UDP-GlcNGc, which isthen used to modify proteins withO-GlcNGc. The kinetics of incorporation and removal ofO-GlcNGc in cells occur in adynamic manner on a time frame similar to that ofO-GlcNAc.Enzymatic activity ofO-GlcNAcase (OGA) toward a GlcNGcglycoside reveals OGA can process glycolyl-containing sub-strates fairly efficiently. A bacterial homolog (BtGH84) of OGA,from a human gut symbiont, also processesO-GlcNGc sub-strates, and the structure of this enzyme bound to a GlcNGc-derived species reveals the molecular basis for tolerance andbinding of GlcNGc. Together, these results demonstrate thatanalogs of GlcNAc, such as GlcNGc, are metabolically viablespecies and that the conserved active site pocket of OGA likelyevolved to enable processing of mis-incorporated analogs ofO-GlcNAc and thereby prevent their accumulation. Such plas-ticity in carbohydrate processing enzymes may be a generalfeature arising from inaccuracy in hexosamine metabolicpathways.