Abstract The amino-terminal fragment, comprising the first 24 residues, has been isolated from rat and human serum albumins after limited peptic digestion. The complete sequence of each fragment has been deduced from the tryptic peptides alone by taking advantage of overlapping peptides resulting from incomplete cleavage. A comparison of the structure of the (1–24) peptides of rat and human albumins with that obtained from bovine serum albumin (J. Biol. Chem., 242, 5451 (1967)) indicates a high degree of similarity. The rat fragment differs from the bovine at three sites, whereas the human fragment differs at four sites. There is a single replacement at Residue 2 common to the rat and human peptides. Because production of this fragment by limited proteolysis must be indicative of certain conformational parameters in the region of Residue 24, it may be concluded that the rat, human, and bovine serum albumin molecules are closely homologous in primary and three-dimensional structure in the amino-terminal region.