Human urine contains growth factors; their physiological roles have not been established. The effect of normal human urine was examined in vitro on clonal growth of human bladder cancer cell lines. Clonal growth of HT-1376, HT-1197, and T24 was enhanced by five different fresh human urine samples from young men. Colony stimulating activity was detected in fractions with a molecular weight greater than 5000 by ultrafiltration. Sephadex G-50 gel chromatography identified two peaks of colony stimulative activity in HT-1376 with molecular weights of approximately 6000 and greater than 12,400, respectively; these two peaks also possessed immunoreactive epidermal growth factor (EGF) and NRK-49F transforming activities. The three bladder cancer cell lines possessed large quantities of EGF specific binding sites and exogenous EGF stimulated colony formation; EGF concentrations in human urine were found to be remarkably higher than those of exogenously added EGF which stimulated clonal growth of bladder cancer cell lines. Moreover, it was demonstrated that fresh urine samples (5%) incubated with anti-human EGF monoclonal antibody (KEM-10) neutralized completely the colony stimulating effects in HT-1376. These results indicate that fresh human urine stimulates clonal growth in human bladder cancer cell lines and that a major part of the activity is represented by urinary EGF. The data promote urinary EGF as a progressive agent of human bladder cancer.