Keratin polypeptides 8 and 18 (K8/18) are intermediate filament phosphoglycoproteins that are expressed preferentially in glandular epithelia. We previously showed that K8/18 phosphorylation occurs on serine residues and that K8/18 glycosylation consists of O-linked single N-acetylglucosamines (O-GlcNAc) that are linked to Ser/Thr. Since the function of these modifications is unknown, we sought as a first step to identify the precise modification sites and asked if they play a role in keratin filament assembly. For this, we generated a panel of K18 Ser and Thr-->Ala mutants at potential glycosylation sites followed by expression in a baculovirus-insect cell system. We identified the major glycosylation sites of K18 by comparing the tryptic 3H-glycopeptide pattern of the panel of mutant and wild type K18 expressed in the insect cells with the glycopeptides of K18 in human colonic cells. The identified sites occur on three serines in the head domain of K18. The precise modified residues in human cells were verified using Edman degradation and confirmed further by the lack of glycosylation of a K18 construct that was mutated at the molecularly identified sites then transfected into NIH-3T3 cells. Partial or total K18 glycosylation mutants transfected into mammalian cells manifested nondistinguishable filament assembly to cells transfected with wild type K8/18. Our results show that K18 glycosylation sites share some features with other already identified O-GlcNAc sites and may together help predict glycosylation sites of other intermediate filament proteins.