Glycosylation is often regarded as being restricted to proteins confined to the cell surface or within the lumen of intracellular organelles. Here we show that the human RNA polymerase II transcription factor Sp1 bears multiple O-linked N-acetylglucosamine (GlcNAc) monosaccharide residues. The lectin wheat germ agglutinin specifically inhibits the transcriptional activation but not the DNA binding function of Sp1. Furthermore, many other RNA polymerase II transcription factors also bear terminal GlcNAc residues, whereas most nuclear proteins, including RNA polymerase I and III transcription factors tested, do not. In some cases, only a subset of the polypeptide species within a particular family of closely related RNA polymerase II factors appears to be glycosylated. Our findings raise the possibility that O-linked GlcNAc residues play a role in the mechanism or regulation of transcriptional activation of RNA polymerase II.