O-GlcNAcase (OGA) promotes O -GlcNAc removal, and thereby plays a key role in O-GlcNAc metabolism, a feature of a variety of vital cellular processes. Two splice transcripts of human OGA encode "long OGA", which contains a distinct N-terminal O -GlcNAcase domain and a C-terminal histoneacetylferase (HAT) domain, and "short OGA", which lacks the HAT domain. The functional roles of long OGA are only beginning to be unraveled, and the characteristics of short OGA remain almost unknown. We find that short OGA, which possesses O -GlcNAcase catalysis machinery like that of long OGA, exhibits comparative resistance to previously described potent inhibitors of long OGA and lysosomal hexosaminidases, including PUGNAc and NAG-thiazoline, suggesting a role for the HAT domain in O -GlcNAcase catalysis. We also find that alpha-GlcNAc thiolsulfonate (2) is the most potent inhibitor of short OGA yet described (K-i = 10 mu M), and exhibits some degree of selectivity versus long OGA and lysosomal hexosaminidases. In contrast to its mode of inhibition of short OGA, 2 acts as a irreversible inhibitor of long OGA by covalently modifying the enzyme as an S-GlcNAc derivative. Covalent attachment of GlcNAc to the HAT domain of long OGA dramatically changes its properties with respect to enzymatic activity and caspase-3 cleavage.