Modification of Ser and Thr residues by attachment of O-linked N-acetylglucosamine [Ser(Thr)-O-GlcNAcylation] to eukaryotic nuclear and cytosolic proteins is as dynamic and possibly as abundant as Ser(Thr) phosphorylation. Known O-GlcNAcylated proteins include cytoskeletal proteins and their regulatory proteins; viral proteins; nuclear-pore, heat-shock, tumor-suppressor, and nuclear-oncogene proteins; RNA polymerase II catalytic subunit; and a multitude of transcription factors. Although functionally diverse, all of these proteins are also phosphoproteins. Most O-GlcNAcylated proteins form highly regulated multimeric associations that are dependent upon their posttranslational modifications. Evidence is mounting that O-GlcNAcylation is an important regulatory modification that may have a reciprocal relationship with O-phosphorylation and may modulate many biological processes in eukaryotes.