The reversed-phase HPLC retention behavior of D-amino acid replacement sets of an amphipathic model peptide, neuropeptide Y, and corticotropin releasing factor has been studied. The results demonstrate that D-amino acid substitutions destabilized the amphipathic alpha-helix, leading to a decrease of fractional helicity as determined by circular dichroism. The effect is enhanced by substitution of two adjacent D-amino acids and correlates well with a decrease of hydrophobic interaction during reversed-phase HPLC, caused by disturbance of the preferred binding domain of the stationary phase-bound peptide. In contrast, D-amino acid substitutions in nonamphipathic or disordered regions of peptides do not influence the retention time to the same extent. Thus, the "retention profile" that results from plotting the retention time vs the position of the double D-amino acid replacements provides an indication of the presence and location of an amphipathic alpha-helical secondary structure in peptides.