The human hybridoma cell line, B9165, was obtained after fusion of lymphocytes from lymph nodes draining the tumor region in a patient with adenocarcinoma of the colon with the human B-lymphoblastoid cell line WI-L2-729-HF2 (729-HF2). B9165 secretes the human monoclonal antibody, C-OU1 (IgM, kappa). Immunocytochemical and immunohistochemical analysis showed that the antibody bound to a differentiation antigen. Electron microscopy of colonic adenocarcinoma cells, intact tumor and colonic epithelium by the immunogold technique demonstrated that the C-OU1 antibody reacted with a molecule associated with areas of disruption of the intermediate filaments in the cytoplasm of the tumor cells. No reaction was seen with intermediate filaments in normal colonic epithelium. The molecular weight of the antigen was shown to be 43 Kda by SDS-PAGE and Western blotting of tumor extracts, and isoelectric focusing of sonicated extracts demonstrated reaction with molecular species of pI 5.4-6.2. These findings suggest that the C-OU1 antigen is a modified cytokeratin 18. The B9165 cell line has proved to be quite stable, and the antibody is of potential clinical value. Its usefulness for localizing tumors in patients is being investigated.