Monoclonal antibody (mAb) 19A211 identifies a superficial bladder cancer-associated sialylated epitope expressed on a heterogeneous group of glycoproteins. These glycoproteins consist of a series of cytoplasmic glycoproteins ranging from 90 to 140 kDa present in tumor cells and, at a lower level, in normal urothelial cells, and of a membrane glycoprotein of 200 kDa observed in tumor cells only. To further characterize this antigenic system, we took advantage of the high avidity of mAb 19A211 to produce a rabbit polyclonal antibody by immunizing with antigen bound to mAb 19A211 immunoaffinity beads. The resulting polyclonal antibody specifically reacts with the 200-kDa species and not with the other glycoproteins. The biochemical characterization of this 200-kDa tumor-associated antigen showed that it is highly glycosylated (more than 50% w/w) and is anchored to the membrane via a glycosyl phosphatidylinositol link; these properties are shared with the carcinoembryonic antigen (CEA). Further experiments showed reactivity of two mAbs directed against protein epitopes of CEA with the 200-kDa component of 19A211 antigen. However, in immunohistochemistry studies of 29 colon and 23 bladder tumor specimens, no correlation was observed between expression of 19A211 and CEA antigens. Moreover, in RIAs, the intensity of expression of the 19A211 carbohydrate epitope relative to a CEA protein epitope was found to be significantly lower with CEA purified from a colon cancer cell line compared to CEA from a bladder cancer cell line. On the basis of these results, we conclude that the 200-kDa component of 19A211 antigen is a CEA glycoform preferentially expressed by superficial bladder tumors.