Immunohistochemical analysis by indirect immunoperoxidase staining demonstrated that monoclonal antibody (mAb) B1.4 derived from a mouse immunized with a bladder cancer cell line EJ-1 was reactive with a high proportion of high-grade and invasive bladder tumors, but not with the majority of low-grade and superficial bladder tumors, or normal urinary epithelium. Among 71 primary bladder tumors classified by pathological grading, positive stainings were observed in 1 of 34 tumors (3%) of grade 1, 8 of 20 tumors (40%) of grade 2 and 14 of 17 tumors (82%) of grade 3. When the tumors were classified by pathological staging, positive stainings were observed in only 8 of 54 (15%) superficial tumors of stages Ta and T1, but in 15 of 17 (88%) invasive tumors of stages T2 and T3. mAb B1.4 showed restricted positive stainings with normal tissues including renal glomerulus, vascular endothelium, squamous epithelium of esophagus, glandular epithelium of prostate, and epithelium of pancreatic acinar gland and minute duct, while positive stainings were observed in a range of tumor tissues other than bladder tumor. Mixed hemadsorption assays with a panel of cell cultures showed also that the antigen recognized by mAb B1.4 was expressed on a range of tumor cell lines. These findings suggest that the antigen recognized by mAb B1.4 may appear after malignant transformation, and be an indicator of malignant potential of bladder cancer.