Chemical Biology of O-GlcNAc: Enzyme mechanisms to chemical probes. O-linked N-acetylglucosamine (O-GlcNAc) is an abundant form of protein O-glycosylation found in the nucleus and cytoplasm of multicellular eukaryotes. This post-translational modification is found on hundreds of proteins yet is regulated by only two enzymes; O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAc is in some cases reciprocal to serine/threonine phosphorylation and has, accordingly, been implicated in essential biological processes. Inappropriate regulation of O-GlcNAc has been proposed to play roles in diseases ranging from cancer to Alzheimer's. Here I discuss our research focused on understanding the substrate specificities and catalytic mechanisms of OGT and OGA, as well as the exploitation of these insights to design probes of these enzymes that are active in cells and tissues. I will also discuss how these tools have enabled us to interrogate the functional roles of O-GlcNAc in various biological models including transgenic Alzheimer mice.