Monoclonal antibodies (mAbs) were developed against corneal endothelial cells (CECs) using cultured bovine CECs as immunogens by the mouse hybridoma technique. One of these mAbs, KP14D10 (IgM type), reacted specifically with CECs in human, bovine and rabbit ocular tissues as judged by immunohistochemical methods. This mAb also reacted with some epithelial cells of human esophageal and gastric glands but not with those of bovine and rabbit origin. The molecular weight of the antigen was determined to be 60,000 Da by the immunoblotting method. This antigenicity was apparently decreased by treatment with hyaluronidase, trypsin, and pronase. In the immunohistochemical study of rabbit fetal corneas, the immunofluorescence appeared specifically in the CECs after the 15th day of gestation and its intensity became stronger as gestation advanced. These results suggest that this antibody would be a valuable tool in the further analysis of corneal development and of divese pathological alterations of CECs.