Epidermal growth factor (EGF) binds with high affinity to the EGF receptor, also known as ErbB-1, but upon replacement of the N-terminal linear region by neuregulin (NRG) 1 or transforming growth factor (TGF) alpha sequences it gains in addition high affinity for ErbB-2/ErbB-3 heterodimers. However, these chimeras weakly bind to ErbB-3 alone. To further dissect the ligand binding selectivity of the ErbB network, we have applied the phage display technique to examine the role of the linear N-terminal region in EGF for interaction with ErbB-2/ErbB-3 heterodimers. A library of EGF variants was constructed in which residues 2, 3, and 4 were randomly mutated, followed by selection for binding to intact MDA-MB-453 cells that overexpress ErbB-2 and ErbB-3 but lack ErbB-1. Analysis of the selected phage EGF variants revealed clones with high binding affinity to ErbB-2/ErbB-3 while maintaining high affinity to ErbB-1. In these variants, Trp (or alternatively His) was almost exclusively present at position 2, while specific combinations of hydrophobic, basic, and small residues were found at positions 3 and 4. The mitogenic activity of the phage EGF variants corresponded with their relative binding affinity. Two of the selected EGF variants, EGF/WVS and EGF/WRS, were further characterized as recombinant proteins. In contrast to previously characterized chimeras of EGF with NRG-1 or TGF-alpha, these variants did not only show high binding affinity for ErbB-2/ErbB-3 heterodimers but also for ErbB-3 alone. These data show that the linear N-terminal region of EGF-like growth factors is directly involved in binding to ErbB-3.