Solubilized epidermal growth factor receptor (EGF-R) has been used in an extension of the Geysen epitope mapping protocol in order to provide additional insight into the amino acid residues in human transforming growth factor alpha (hTGFalpha) which are critical to recognition and binding. Overlapping heptapeptides which encompassed the 50 amino acid primary sequence of hTGFalpha were synthesized on a polyethylene solid phase, and the amount of detergent-solubilized EGF-R bound to each peptide was measured using ELISA. EGF-R appeared to bind reproducibly to four heptapeptides cognate to sequences in both the N- and C-domains of hTGFalpha (residues 22-28, 28-34, 36-42, and 44-50). Visualization of these four regions on three-dimensional solution phase structures of hTGFalpha, derived from H-1 NMR measurements [Kline, T.-P., Brown, F. K., Brown, S. C., Jeffs, P. W., Kopple, K. D., & Mueller, L. (1990) Biochemistry 29, 7805-7813], indicated that the peptide segments are located on a single face of the protein and suggested the presence of a potential receptor binding cavity. If peptide segments within both the N- and C-domains of hTGFalpha are involved in binding to EGF-R, then this has direct consequences for possible molecular mechanisms by which receptor activation might take place. For example, the observed conformational flexibility in the six NMR-derived hTGFalpha structures due to variations in the main-chain torsion angles of Val-33, in combination with the involvement of residues from both domains in the proposed binding cavity, may imply that receptor activation results from interdomain reorientation in the protein ligand. Such a model is consistent with recent investigations of the interaction of EGF-R and its ligands using physical methods, which have indicated changes in the solution conformation of the receptor upon ligand binding [Greenfield, C., Hiles, I., Waterfield, M. D., Federwisch, M., Wollmer, A., Blundell, T. L., & McDonald, N. (1989) EMBO J. 8, 4115-41231. We anticipate that the receptor-binding assay reported in this study might also be more generally applicable in probing the interaction of other biologically important peptides, and proteins, with cellular receptors of similar structure to that of EGF-R.