We have examined the resolution, on reversed-phase columns, of a series of model synthetic peptides and commercially available synthetic peptide standards under gradient elution conditions, using a water-acetonitrile mobile phase containing hydrophilic (phosphoric acid) or hydrophobic (trifluoroacetic acid, heptafluorobutyric acid) ion-pairing reagents. Increasing hydrophobicity or concentration of the ion-pairing reagents increased peptide retention times. It was clearly shown that these reagents effected changes in peptide retention time solely through interaction with the basic residues in the peptide. In general, each positive charge, whether originating from a lysine, arginine or histidine side-chain, or from an N-terminal alpha-amino group, exerts an equal effect on peptide retention. Different counterions have different effects on the change in peptide retention time per positively charged residue due to their differences in hydrophobicity. However, increasing concentrations of a specific counterion have an essentially equal effect per positively charged residue. These effects are also column dependent (n-alkyl chain length and ligand density). These results, demonstrating a simple relationship between peptide retention in different ion-pairing systems, enabled the determination of rules for prediction of peptide retention times in one ion-pairing system from observed or predicted retention times in another system. The small average deviation of predicted and observed retention times for a series of basic peptides was good evidence for the value of this predictive method. This study provides a clear understanding of the effect of changing counterion hydrophobicity or concentration on peptide retention, and thus can be extremely beneficial in the purification of peptides and for providing proof of peptide homogeneity.