A monoclonal antibody has been produced that binds to the apical squames (flattened cells) of the rat ocular surface epithelium and to the goblet cells of the conjunctiva. Immunoelectron microscopic localization of the antigen indicates that in apical cells it is present along the apical-microplical membrane in the region of the glycocalyx. In subapical squames, the antigen is in cytoplasmic vesicles. In some goblet cells, the antigen is in the Golgi network, and in others, it is located primarily in the membrane of the mucous granules. SDS-PAGE and immunoblot analysis demonstrate that the molecular weight of the antigen is greater than 205 kD, and the electrophoretic band stains with Alcian blue followed by silver stain. Periodate oxidation of immunoblots and cryostat sections removes antibody binding. Neuraminidase treatment of cryostat sections does not remove antibody binding, whereas N-glycanase does. Taken together, these data indicate that the antigen recognized by the monoclonal antibody is a carbohydrate epitope on a high-molecular-weight, highly glycosylated glycoprotein in the glycocalyx of the ocular surface epithelium and goblet cell mucin granule membrane. The antigen appears to be stored within cytoplasmic vesicles and reaches the glycocalyx when cells differentiate to the apical-most position where the glycocalyx interfaces with the mucin layer of the tear film.