A sensitive and simple two-site enzyme immunoassay was developed for rat epidermal growth factor (rEGF), which was purified from male rat submaxillary glands. This system is based on the sandwiching of the antigen between anti-rat EGF IgG antibody coated on a polystyrene bead and anti-rat EGF Fab' antibody-linked peroxidase, whose activity was stable at 4 degrees C for 6 months. In this system, the rat EGF was detectable at a concentration of 2 pg/tube. No interference was observed by addition of human plasma. There was no cross-reactivity with mouse EGF, human EGF, or other substances with similar chemical structures.