Several chiral thiols, i.e. traditionally used enantiomerically pure SH reagents and novel N-R-mandelyl-L-cysteine (R-NMC) containing additional chiral center, have been applied as co-reagents in precolumn derivatization with o-phthalaldehyde for enantiomeric HPLC analysis of individual alpha-amino acids and their mixtures. The R-NMC-derived isoindoles as well as adducts with other thiols have a characteristic absorption maximum at 340 nm, and are highly fluorescent allowing detection of 10 microg/l of an amino acid. Investigated 19 amino acids were analyzed separately and in a mixture by a gradient HPLC after precolumn derivatization. The chromatographic behavior of formed isoindoles substantially differs for each of the thiols used for modification. In contrast to traditional enantiomeric thiols application of diastereomeric R-NMC provides higher resolution for alpha-amino acid enantiomers, with L,D-elution order (except for Arg). Combined use of R-NMC and other thiol enlarges the possibilities of this method, allowing accurate chiral analysis of complex amino acid mixtures.