The physicochemical relationships of four new scales of amino acid hydrophobicity coefficients, derived from the reversed-phase high-performance liquid chromatographic (RP-HPLC) retention data of 1738 peptides, have been compared with 12 previously published scales of amino acid hydrophobicities. Four different reversed-phase chromatographic systems were used to obtain the experimental data, including three well-characterized hydrophobic adsorbents of different n-alkyl ligand chain length (n-octadecyl (C18), n-octyl (C8), and n-butyl (C4) ligands) and two different aquo-organic eluents, namely a binary water-acetonitrile and a ternary water-acetonitrile-2-propanol elution system, both containing 0.1% trifluoroacetic acid. Significant correlations were observed between the amino acid hydrophobicity scales derived from the RP-HPLC data of these peptides separated with the C18- or C8-silica adsorbents and the water-acetonitrile elution system and the hydrophobicity scales based on the transfer free energies of an amino acid entity from a polar to a nonpolar solvent or the statistical degree of exposure to the surrounding solvent of an amino acid side chain within a protein structure. Conversely, lower levels of correlation were observed between the previously calculated hydrophobicity scales for the common a-amino acids and the amino acid hydrophobicity coefficient values derived from this peptide structure-retention data base with the C4 ligand or the C18 ligand in combination with the water-acetonitrile-2-propanol solvent system. The results confirm that the relative ranking and magnitude of the hydrophobicities of amino acid side chains within polypeptides are dependent upon the chemical microenvironment of the interface established between the solute, the solvent, and the immobilized hydrocarbonaceous ligand. The availability of this extensive data base of peptide structure-chromatographic retention behavior has also permitted differences in peptide-nonpolar ligand interactions to be quantified in physicochemical terms, including the propensity of amino acid side chains in peptide structures to partition into different hydrophobic environments from aqueous solutions of different hydrogenbonding or dipolar characteristics.