The enantiomeric purity of the pharmaceutical D-penicillamine (I) has been determined by a novel high-performance liquid chromatographic technique involving the formation of 5,5-dimethylthiazolidine-4-carboxylic acid (III) by reaction of I with formaldehyde and separation of the optical antipodes by means of ligand exchange chromatography using the copper(II) complex of (2S,4R,2'RS)-4-hydroxy-1-(2'-hydroxydodecyl)proline, which is coated on a reversed-phase column. The limit of determination for the L-antipode is ca. 0.1%. The validation of the method is accomplished by comparison with an independent gas chromatographic procedure. Thirteen commercially available lots of I are shown to be of equally high enantiomeric purity (ca. 99.9%).