The binding and internalization of epidermal growth factor (EGF) in human epithelioid carcinoma cells (A-431), which have approximately 2.6 X 10(6) receptors per cell, has been followed with 125I-labeled EGF and by fluorescence microscopy. We have prepared a fluorescent derivative of EGF that is biologically active and retains substantial binding affinity for cell receptors. After binding of this derivative to cells at 6 degrees, the cellular borders were prominently stained and the fluorescence on the remainder of the membrane was uniform. Upon warming of these cells to 37 degrees for 10 min, the surface fluorescence diminished and randomly distributed endocytotic vesicles appeared in the cytoplasm. After 20 min at 37 degrees these fluorescent vesicles formed a perinuclear ring. The binding of EGF to the surface of these cells was also visualized by immunofluorescence using rabbit antibodies to EGF and rhodamine-labeled goat anti-rabbit antibodies. We did not detect large fluorescent clusters or cap formation in these experiments. These data provide direct confirmation of the previous biochemical data that suggested that cell membrane-bound EGF is rapidly internalized.