Transforming growth factor alpha-Pseudomonas exotoxin-40 (TP40) is a hybrid fusion protein that selectively binds to cancer cells that express the epidermal growth factor receptor. TP40 is then internalized and kills these cells by virtue of its Pseudomonas exotoxin-derived domains. We studied the safety and short-term antitumor activity of intravesical TP40 in 43 patients with refractory superficial bladder cancer. These patients had resected Ta/T1 disease (n = 19), visible Ta or T1 lesions (n = 11), or carcinoma in situ (n = 13). Patients were treated with increasing dose levels of TP40 at 0.15, 0.3, 0.6, 1.2, 2.4, 4.8, or 9.6 mg/week for 6 weeks and evaluated by comparing pretreatment and posttreatment cystoscopic examinations, cytology, and histopathology. All TP40 doses were well tolerated. No evidence of antitumor activity was seen in any of the patients with Ta or T1 lesions. However, 8 of 9 patients with evaluable carcinoma in situ were judged by histopathology of multiple biopsy specimens to exhibit clinical improvement following TP40 therapy. In most of these responsive patients, cystoscopic examination supported the histopathological findings, although cytology of urine and bladder washings persistently demonstrated malignant cells. Therefore, TP40 appears to be a well-tolerated biological agent that may prove to have utility in treating carcinoma in situ of the bladder.