A primary receptor-binding region of mouse epidermal growth factor (EGF) was identified by comparing the relative affinities of selected synthetic fragments with overlapping sequences in the EGF receptor-binding assay, using human foreskin fibroblasts. Only synthetic peptides containing the amino acid residues 20-31 in the mouse EGF sequence showed the ability to compete with 125I-labeled EGF in binding to EGF receptors. The affinities of the cyclic EGF fragment [Ala20]EGF-(14-31) and the linear [(S-acetamidomethyl)-Cys20,31]-EGF-(20-31) were approximately 1/10(4) of the affinity of EGF. Despite their reduced receptor affinities, these two peptides exhibited the in vitro biological activities of native EGF, while fragments from other regions of the EGF molecule were devoid of these biological properties. The peptides induced DNA synthesis in human foreskin fibroblasts as measured by [3H]thymidine incorporation into DNA. They also induced EGF receptor clustering and activated the EGF-sensitive kinase, enhancing the autophosphorylation of EGF receptors in a dose-related manner. Moreover, a major antigenic determinant of EGF for rabbit anti-EGF antibodies was identified within this same localized region of the EGF molecule by competition experiments utilizing the synthetic EGF fragments. The predominant EGF antigenic determinant(s) was also found within the fragment [(S-acetamidomethyl)Cys20,31]-EGF-(20-31). The accessibility of the residues in positions 20-31 for antibody recognition is consistent with the conclusion that these residues constitute or contain a major receptor-binding region for EGF.