Abstract The separation of d- and l-enantiomers of 6-(polyhydroxypropyl)pterins was obtained by ligand-exchange chromatography using a reversed-phase column at 12°C with a mobile phase containing d-phenylalanine as the chiral modifier and Cu(II) as the metal ion. This allowed the determination of the stereoconfiguration of natural pterins from very small amounts of biological sample containing pterins in the picomole range (nanogram range). Fluorescence detection was used both to increase the sensitivity and to confirm the identification by on-line fluorescence spectroscopy and comparison with reference compounds. The stereoconfiguration of optically active pterins present in a bacterium ( Escherichia coli), in a ciliate protozoan ( Tetrahymena pyriformis), in an amoeba ( Dictyostelium discoideum), and in mammals (human urine) was obtained and compared to earlier determinations. Incidental findings resulting from the application of this method were that human urinary monapterin and the major pterin of T. pyriformis were identified as a d-monapterin, which, until now, was not known as a natural pterin.