The loss of blood group isoantigens from the surface of bladder tumor cells has been correlated with the potential invasiveness of the tumor. Development of simple and reliable methods for detection of these isoantigens should facilitate the general clinical use of this test for predicting malignant potential in low grade, low stage cancer of the bladder. We now report a direct peroxidase technique for the detection of isoantigens A and B by utilizing the specific interaction between biotin and avidin, and the capability of labeling a single antibody with multiple biotin molecules. Antibodies specific to the isoantigens A and B were purified from human antisera by affinity chromatography using an immunoabsorbent containing chemically synthesized antigenic determinants. The purified antibodies were directly labeled with biotin. An avidin-biotin-peroxidase complex was used to bind the biotinylated antibody for the peroxidase staining reaction of the isoantigens on tissue section. Application of this technique to formalin-fixed, paraffin-embedded bladder tissue and tumor sections yielded specific and strong stainings of the isoantigens with low background staining. The potential clinical application of this method requires further evaluation.