Twenty-one analogues of ACTH, three analogues of LH-RH and four insulins have been successfully separated on a commercial reversed-phase material with tartrate buffer--acetonitrile systems containing sodium 1-butanesulphonate and sodium sulphate as the mobile phase. The effect of the constituent amino acid residues on the order of elution has been studied in detail by using a variety of closely related peptides; the order of elution of a series of peptides, which differ by only one amino acid residue, can in most instances be explained in terms of the difference in the hydrophobicities of the amino acid residues concerned, but in some instances, such as in diastereoisomers or positional isomers, the order of elution must be interpreted in terms of the hydrophobicity of the whole peptide molecule. This chromatographic method has been proved to be very useful for the rapid examination of the purity of these peptide hormones and for the separation of closely related peptides with molecular weights up to ca. 6000.