Drosophila polytene chromosomes contain a surprisingly large amount of terminal N-acetylglucosamine (GlcNAc) residues along their lengths, as determined by staining with a fluorescently tagged lectin, wheat germ agglutinin (FITC-WGA) and by specific radio-labeling with bovine galactosyltransferase and UDP-[3H]galactose. FITC-WGA intensely stains polytene chromosomes in a distinctive banding pattern in which condensed chromatin is brightly labeled and transcriptionally active "puff" regions are less intensely stained. Biochemical analyses of galactosyltransferase-radiolabeled chromatin indicates that nearly all of the chromatin-associated GlcNAc moieties exist as single monosaccharide residues attached to protein by an O-linkage (O-GlcNAc). Chromatin is enriched in O-GlcNAc (over 400 pmol/micrograms of chromatin protein) as compared with total nuclei and other cellular compartments. O-GlcNAc moieties are found on a myriad of chromatin proteins that have diverse types of intermolecular associations with other nuclear components.