The solution structure of the 53 amino acid peptide hormone, human epidermal growth factor (hEGF), has been determined to high resolution from nuclear magnetic resonance (n.m.r.) data. A large number of internuclear distance and dihedral restraints was obtained, including data from uniformly 15N-labelled hEGF. Dynamical simulated annealing methods using the program XPLOR were used for structure calculation. An improved protocol was developed combining efficient conformational searching at a reduced computational cost. The general fold of the calculated structures compared well with that of a derivative of the carboxy-terminally truncated hEGF determined previously. A group of 44 structures were calculated with no violations greater than 0.3 A and 3 degrees for distance and dihedral restraints, respectively. The average pairwise root mean square (r.m.s.) deviation of all backbone atoms for these structures was 2.25 A for all 53 residues, 0.92 A for the bulk of the protein, and 0.23 A for the functionally important carboxy-terminal domain. Two new helical segments containing highly conserved amino acids have been identified; one between cysteines 6 and 14 and a second at the end of the carboxy-terminal domain. New insight into the molecular architecture of the site of putative receptor binding was provided by comparing the structure of hEGF with its biologically equipotent analogue, human transforming growth factor alpha. This comparison revealed a close structural relationship between the two growth factors and provides an improved understanding of the structure/function relationships in EGF.