Although the knowledge that nuclear and cytoplasmic proteins are modified with N -acetylglucosamine has existed for decades, little has been shown as to its function until recently. There are now substantial data highlighting the significance of proper regulation of this modification in multiple cellular processes. Currently, only two enzymes are known that regulate this modification. O -GlcNAc transferase (OGT) modifies protein substrates posttranslationally by adding the N -acetylglucosamine. Bifunctional nuclear/cytoplasmic O -GlcNAcase and acetyl transferase (NCOAT) is responsible for cleaving the modification from target proteins. Here, we demonstrate for the first time an unusual association of these two opposing enzymes into a single O -GlcNAczyme complex. NCOAT and OGT associate strongly through specific domains such that NCOAT accompanies OGT, with histone deacetylases (HDACs), into transcription corepression complexes. Exclusion of NCOAT activities from OGT association blocks proper estrogen-dependent cell signaling as well as mammary development in transgenic mice. This demonstrates that NCOAT is in a strategic position to rapidly counteract OGT and HDAC without requiring its recruitment. © 2006 Oxford University Press.