A mAb T2-2 was generated using hybridoma techniques, and its target was identified as a 46 ku-cytokeratin (CK), based on biochemical study and a completely overlapped binding pattern of mAb T2-2 with anti-pan-CKs antibodies. An epithelia-specificity of the mAb T2-2 was determined by screening 68 human normal and 65 tumor tissues using immunohistochemistry. Unlike most of anti-CKs antibodies, the mAb T2-2 recognized a mono-specific epitope only expressed on the 46 ku CK, suggesting that mAb T2-2 is superior to most anti-CKs antibodies that cross-reacted with many different kinds of CKs. In addition, it was found that the mAb T2-2 was multipurpose with a broad applicability to ELISA, immunohistochemistry, immunofluorescence, Western blotting, and was also compatible with various fixation reagents. These results strongly indicate that the mAb T2-2 has potential applications for studying CKs function and for diagnosis of tumor and other disorders.