Aromatic amino acid residues in epidermal growth factor (EGF) isolated from the rat have been investigated by proton n.m.r. and nuclear Overhauser methods at 500 MHz and by photochemically induced dynamic nuclear polarization (photo-c.i.d.n.p.) experiments at 360 MHz. Rat EGF contains six aromatic residues, i.e. one histidine and five tyrosine residues. pH titration data allow identification of the histidine imidazole ring protons, whereas two-dimensional n.m.r. correlated spectroscopy establishes connectivities between tyrosine ring (2,6) and (3,5) proton resonances. Photo-c.i.d.n.p. data give evidence for solvent exposure of the one histidine and the five tyrosine residues in rat EGF. Nuclear Overhauser experiments and pH titration data suggest proximity relationships among four of the tyrosine residues and the histidine residue. These data indicate the presence of a clustered, aromatic, structural domain on the protein surface and may provide a clue to the understanding of the functional structure of EGF.