Murine epidermal growth factor (EGF), a 53 amino acid protein, has been modified by enzymic digestion, site-specific chemical reactions, and recombinant DNA technology. After trypsin digestion the EGF derivatives EGF1-48(called EGF-T) and EGF1-45(called EGF-T2) were separated from the residual EGF and the C-terminal pentapeptide by reversed-phase high-performance liquid chromatography. EGF-T competes for binding to EGF receptors with the same efficiency as EGF. the EGF-T2derivative had no detectable receptor binding activity even at 100 nM. the in vitro mitogenic potencies of EGF and EGF-T for Balb/c 3T3 cells were indistinguishable. Treatment of EGF-T with carboxypeptidase Y yielded two derivatives, EGF-T-(des-Arg48) and EGF-T-des(Leu47-Arg48). There was only a 3-7-fold diminution in the binding efficiency and mitogenic potency for EGF-T-(des-Arg48). However, there was more than a 100-fold decrease in the binding efficiency and mitogenic activity of EGF-T-des(Leu47-Arg48). These results indicated that Leu47is intimately involved in the formation of the ligand-receptor complex. Studies with a number of proteases indicated that the C-terminus of EGF was susceptible to enzymic digestion; however, the N-terminus appears to be folded into a conformation which prevents access to proteolytic digestion. Consequently, the N-terminus was modified by preparing an analogue with recombinant DNA technology. Oligonucleotides corresponding to EGF(3_48).Met3.Lys21residues were ligated in frame to a β-galactosidase expression vector. the β-Gal-EGF fusion protein was cleaved with cyanogen bromide and EGF(4_48).Lys21purified. This derivative was equipotent with EGF in the mitogenesis assay and bound to the EGF receptor with the same affinities as EGF. Disruption of the central antiparallel β-sheet structure of EGF at Met21by treatment of EGF with cyanogen bromide reduced both the binding efficiency and the mitogenic activity of EGF more than 100-fold.