The extracellular matrix adhesion molecule fibronectin exhibits different isoforms derived by alternative splicing as well as recently demonstrated variation in O-glycosylation. Although fibronectin is widely distributed in normal tissues, the individual isoforms have been found to show restricted tissue distribution and association with malignancies. The monoclonal antibody FDC-6 defines a cancer-associated de novo glycosylation of a specific threonine residue in the C-terminal region of the fibronectin molecule termed oncofetal fibronectin. Here we report an immunohistological study of oral squamous cell carcinomas (n = 33), premalignant lesions (n = 15), and normal oral mucosa (n = 10) using the FDC-6 antibody. A selective expression of the oncofetal fibronectin epitope was demonstrated in close relation to the invading carcinoma, whereas no staining was observed in premalignant lesions without epithelial dysplasia, or in normal epithelium. Furthermore, we attempted to identify additional carbohydrate-related epitopes distinguishing fibronectin of human hepatoma cell line HUH-7 from plasma fibronectin. No novel epitopes were identified, as all generated monoclonal antibodies lacking reactivity with plasma fibronectin showed the same specificity as FDC-6. Previous studies have indicated that the de novo glycosylation is induced by a novel transferase activity only found in fetal and carcinoma cell lines, placenta and hepatoma tissues. Here we provide further evidence that a purified UDP-GalNAc:peptide N-acetylgalactosaminyltransferase from normal bovine thymus and human placentae is incapable of utilizing the hexapeptide VTHPGY as a substrate. The results demonstrate that oncofetal fibronectin is highly associated with malignancy, and appears to be induced by expression of a unique glycosyltransferase or modification of the specificity of the normally expressed transferase.