We report on a novel posttranslational modification of cytoplasmic proteins. Presented evidences suggest that cytokeratins are bound in vitro by mammalian galectin-3 and the galectins from the sponge Geodia cydonium via their type II carbohydrate recognition domains, whose highest binding affinity is directed towards terminal alpha-N-acetylgalactosamine-bearing glycans with the general sequence GalNAcalpha1-3Gal(NAc)beta. Specificity analyses and the characterization of the critical sugar residue on cytokeratins for galectin binding were done with cytochemical and biochemical methods using various plant and animal lectins. Binding of GalNAc-specific lectins was saturable, sensitive to mild periodate oxidation, inhibitable by glycoconjugates carrying terminal GalNAc, and abolished after treatment of the cytokeratins with alpha-N-acetylgalactosaminidase. Binding to bacterially expressed recombinant cytokeratins did not exceed background binding. The presence of GalNAc residues on highly purified cytokeratins from MCF-7 and HeLa SS6 cells was confirmed by sugar composition analyses using gas chromatography/mass spectrometry. This novel posttranslational modification was not restricted to cytokeratins of MCF-7 cells, but did also occur in all of 9 other examined human carcinoma cell lines and in a normal human mammary epithelial cell line. From these cytochemical and biochemical in vitro studies we hypothesize that this glycan with its terminal alpha1-3 linked GalNAc determinant might represent the first natural cytoplasmic ligand for endogenous galectins-3 detected so far.