A human cancer cell line (ZK-1) has been established from a well-differentiated squamous cell carcinoma of the tongue. The cytokeratin polypeptides pattern of ZK-1 cells consists of four major polypeptides with molecular weights between 46 and 58 kilodaltons (kd). Antibodies raised against the purified 58 kd cytokeratin filament from cultured ZK-1 cells were shown to be specific by one- and two-dimensional polyacrylamide gel electrophoresis, immunofluorescence, and immunoelectron microscopy. Immunoprecipitation studies showed that the antibody reacted mainly with the 58 kd cytokeratin. The distribution of the 58 kd cytokeratin in both sparse and confluent cultures was analyzed by the indirect immunofluorescence technique. In both cases, it appeared that fibrillar arrays extended throughout the cytoplasm running over the nucleus and toward cell-to-cell boundaries. Thick bundles of filaments were seen in confluent cultures, especially in large and flattened keratinized cells. Characteristically, reactions were also seen in the intercellular boundaries, appearing as "dots." Electron microscopy using immunoperoxidase techniques indicated that the 58 kd cytokeratin was localized in tonofilaments, tonofilaments attached to desmosomes, desmosomal plaques and membrane-coating granules.