A mouse IgG1 monoclonal antibody (MAb), 19A211, defining a tumor-associated cell-surface antigen of superficial papillary bladder tumors, was generated by immunizing with fresh bladder tumor cells mice neonatally injected with normal human urothelial cells. The reactivity of the antibody with cell lines was examined by indirect immunofluorescence staining and was restricted to 3/14 bladder cancer lines and 3/31 cancer cell lines of non-bladder origin, including HeLa cervical cancer. No normal fibroblast, kidney cells, EBV-lymphocytes, erythrocytes or leukocytes expressed the antigen. Reactivity of MAb 19A211 was well preserved on tissue paraffin sections. Immunoperoxidase staining of normal adult or fetal tissues showed no reactivity except for a patchy or uniform staining of umbrella cells in 6/23 adult and 1/4 fetal urothelium samples. Positive and often heterogeneous staining was observed on 24/38 papillary superficial tumors (Ta) and 4/5 carcinoma in situ bladder lesions but on only 4/20 infiltrating tumors. It was also observed on 5/6 cervical condylomas and one bladder condyloma, but none of 6 penile or vulvar condylomas. All other tumors tested were negative. The antigenic determinant is present on a heterogeneous group of proteins with molecular weights ranging from 90 to 200 kDa. It is sensitive to periodate treatment and to neuraminidase but only partially sensitive to proteases. MAb 19A211 is different from other reported MAbs with similar reactivity to superficial bladder tumors and umbrella cells of normal urothelium. When tested in competition assays, several of these MAbs, but not 19A211, were found to react with Lewis X blood group determinant. Our results suggest that 19A211 may be useful for detection and stratification of bladder tumors.