Many proteins contain so-called epidermal growth factor (EGF)-like domains that share the characteristic spacing of cysteines and glycines with members of the EGF family. They are, however, functionally unrelated, despite the fact that the three-dimensional structure of these EGF-like domains, also, is often very similar to that of the EGF receptor agonists. In the present study, we linked an EGF-like repeat from the Drosophila Notch protein to the N-and C-terminal linear tail sequences of human EGF (hEGF), and we showed that this chimera (E1N6E) is unable to bind or activate the hEGF receptor. This recombinant protein was then used as a basic construct for identifying the minimal requirements for high affinity EGF receptor binding and activation. We selectively reintroduced a limited number of important hEGFderived residues, and by using this unique approach, we were able to make hEGF/Notch chimeras that, compared with wild type hEGF, showed nearly 100% binding affinity and mitogenic activity on HER-14 cells expressing the hEGF receptor.Polypeptide growth factors acting through their tyrosine kinase receptors play a central role in the outgrowth of tumors. Many tumor cells are able to secrete such growth factors, to which they can respond in an autocrine fashion. High expression of members of the epidermal growth factor (EGF) 1 family, in particular of transforming growth factor ␣ (TGF␣), has been found in a variety of human cancers including breast and prostate cancers; oral and head and neck cancers; tumors of the gastrointestinal tract; and colon, lung, liver, kidney, and ovarian cancers (1-4). Because most of these tumors also express the EGF receptor, there is evidence that TGF␣ cause autocrine growth stimulation in these tumors. In order to interfere with such autocrine processes, it might be of great interest to develop an EGF receptor antagonist, an EGF-like protein with high receptor binding affinity that is, however, unable to activate the receptor. Although attempts to design such an antagonist have as yet been unsuccessful, much progress has been made in elucidating the requirements for interaction of EGF with its receptor.