To assist in the description of the cellular heterogeneity present in normal and neoplastic urothelium, a panel of monoclonal antibodies (MoAbs) was raised against human transitional cell carcinoma (TCC) of the urinary bladder. All immunizations were carried out using whole cells and membrane preparations from well differentiated human TCCs. Two fusions produced 145 hybridomas. Following primary screening by ELISA and secondary screening with immunohistochemistry, three useful antibodies were identified. MoAb 35.48 binds to all cell layers of the normal urothelium and well differentiated tumours, but not to the majority of poorly differentiated tumours. MoAb 21.48 binds preferentially to the basal cell layer of normal urothelium and to some well differentiated papillary TCCs, but poorly differentiated tumours exhibit diffusely positive staining. MoAb 21.48 also shows cross-reactivity with basal cell layers of other epithelia. MoAb 5.48 binds preferentially to the superficial cell layers of normal urothelium and well differentiated TCCs, but exhibits less binding in poorly differentiated tumours with loss of the preferential superficial staining. Quantitative flow cytometric studies indicate that MoAb 5.48 binds to a cell-surface antigen which is present on significantly fewer cells of poorly differentiated tumours than on either normal urothelium (P less than 0.05), or well differentiated tumours (P = 0.05).