Hybridoma antibodies to the human bladder cancer cell line RT4 were prepared by fusing spleen cells from RT4-immunized BALB/c mice with the murine plasmacytoma line NS-1. When antibodies were characterized by direct serological testing, antibody A2 exhibited highly restricted specificity for an antigen found only on RT4. However, further analysis with absorption, a standard serologic technique that has not been widely applied to hybridoma antibodies because of their monoclonal nature, revealed that the antigen could also be found on 1 other bladder cancer cell line, 5637, and on the human cervical carcinoma line ME-180. Quantitative absorption assays suggest that this phenomenon of absorption-positive, direct test-negative cells may be related to the amount of antigen present on the cell surface. Another antibody, A80, detected an antigen with broader distribution. Both antibodies described heat-labile, trypsin-resistant antigens present on a restricted range of cells. The role of these antibodies in identifying subsets of malignant urothelial cells is being investigated.