While attempting to derive a set of high-performance liquid chromatographic amino acid retention coefficients from a set of 298 related peptide analogues of a 13-amino acid peptide, we found that each position within the peptide would require a different set of retention coefficients to accurately predict peptide retention times. Furthermore, our results show that peptides having the same amino composition but slightly different sequences can have very different retention times. We believe that individual sequence domains and the resulting differences in the solid phase-mobile phase interactions must be taken into account for the accurate prediction of peptide retention times.