Addition of epidermal growth factor (EGF) to cultures of the urinary bladder carcinoma cell line 5637 regulated proliferation and production of colony stimulating activity (CSA). The optimal concentration range of EGF for stimulation of cell proliferation was 5-20 ng/ml EGF and for production of CSA 2-20 ng/ml EGF. High EGF concentrations (100-200 ng/ml) showed inhibitory effects on proliferation and to a greater extent on CSA production. Also, EGF binding sites of high affinity (kd:3.25 nM) were demonstrated on the cell surface. In the optimal concentration range for stimulation (5-20 ng/ml EGF) EGF binding sites were occupied half-maximally. The loss in EGF binding after long incubation at 37 degrees C was prevented by the lysosomal inhibitory agent, chloroquine. Nonspecific binding of EGF was very low, the amount of maximally bound EGF was 1430 fmol/mg protein (130,000 bound EGF molecules/cell). A strong band of approximately 170,000 daltons could be detected by means of an anti-erbB serum which recognizes the EGF receptor protein. The protein became phosphorylated upon addition of gamma-32P ATP. The data suggest that EGF initiates its action by binding to specific high affinity receptors and plays a role in growth regulation and differentiation of the urinary bladder carcinoma cell line 5637.