Immunohistochemistry for uroplakin III (UP III), cytokeratin 7 (CK 7), and cytokeratin 20 (CK 20) using commercially available antibodies was done in normal canine urinary bladder and 72 canine urinary bladder tumors that had been fixed in formalin and embedded in paraffin. Prolonged fixation (3-28 days) did not significantly alter the immunostaining for UP III. There was moderate reduction in the intensity for CK 7 and CK 20 after 1 week of fixation. UP III was detected in superficial (umbrella) cells and some intermediate cells of the normal urinary bladder, 7 of 7 transitional cell papillomas (TCPs), 50 of 55 transitional cell carcinomas (TCCs), and 4 of 5 metastatic TCCs. Staining was typically outlined in the plasma membrane, but diffuse or focal cytoplasmic staining was also observed. Intracytoplasmic lumina were usually positive for UP III. One squamous cell carcinoma of the bladder, 4 nonepithelial bladder tumors, and 285 nonurothelial tumors from different nonurinary locations were negative for UP III. CK 7 was detected in 7 of 7 TCPs, 53 of 54 TCCs, and 5 of 5 metastatic TCCs. The staining for CK 7 was diffuse cytoplasmic. CK 20 was detected in 1 of 7 TCPs, 37 of 54 TCCs, and 1 of 5 metastatic TCCs. The staining with CK 20 was cytoplasmic and weaker than with antibodies to UP III or CK 7. There was concurrent expression of UP III, CK 7, and CK 20 in 36 of 54 TCCs. UP III is a specific and sensitive marker for canine transitional epithelial (urothelial) neoplasms, detecting 91% of TCCs. Negative results may be observed with anaplastic tumors.