Copper(II) complexes of L-amino acylamides (Phe, Val, Tyr, Ala) when added to the eluent (water-acetonitrile) in reversed-phase high-performance liquid chromatography (C18) are able to perform enantiomeric separation of dansylamino acids. The lipophilicity and bulk of the ligand greatly affect the stereoselectivity and the elution order of the enantiomers. The type and concentration of the copper complexes, pH and eluent polarity were examined in order to get some insights into the separation mechanism. This may be consistent with a ligand-exchange mechanism, probably occurring on the organic phase of the column, where the enantioselective complex is adsorbed. Mixtures of D,L-dansylamino acids were well separated by isocratic and gradient elution.