The post-translational modification of serine and threonine residues of nucleocytoplasmic proteins with 2-acetamido-2-deoxy-d-glucopyranose (GlcNAc) is a reversible process implicated in multiple cellular processes. The enzyme O-GlcNAcase catalyzes the cleavage of beta-O-linked GlcNAc (O-GlcNAc) from modified proteins and is a member of the family 84 glycoside hydrolases. The family 20 beta-hexosaminidases bear no apparent sequence similarity yet are functionally related to O-GlcNAcase because both enzymes cleave terminal GlcNAc residues from glycoconjugates. Lysosomal beta-hexosaminidase is known to use substrate-assisted catalysis involving the 2-acetamido group of the substrate; however, the catalytic mechanism of human O-GlcNAcase is unknown. By using a series of 4-methylumbelliferyl 2-deoxy-2-N-fluoroacetyl-beta-D-glucopyranoside substrates, Taft-like linear free energy analyses of these enzymes indicates that O-GlcNAcase uses a catalytic mechanism involving anchimeric assistance. Consistent with this proposal, 1,2-dideoxy-2′-methyl-alpha-D-glucopyranoso-[2,1-d]-Delta2′-thiazoline, an inhibitor that mimics the oxazoline intermediate proposed in the catalytic mechanism of family 20 glycoside hydrolases, is shown to act as a potent competitive inhibitor of both O-GlcNAcase (K(I) = 0.070 microm) and beta-hexosaminidase (K = 0.070 microm). A series of 1,2-dideoxy-2′-methyl-alpha-D-glucopyranoso-[2,1-d]-Delta2′-thiazoline analogues were prepared, and one inhibitor demonstrated a remarkable 1500-fold selectivity for O-GlcNAcase (K(I) = 0.230 microm) over beta-hexosaminidase (K(I) = 340 microm). These inhibitors are cell permeable and modulate the activity of O-GlcNAcase in tissue culture. Because both enzymes have vital roles in organismal health, these potent and selective inhibitors of O-GlcNAcase should prove useful in studying the role of this enzyme at the organismal level without generating a complex chemical phenotype stemming from concomitant inhibition of beta-hexosaminidase.