An extensive study of the behavior of three chiral stationary phases (CSP) is presented for the stereoselective determination of methadone. The following chromatographic columns were selected: a cellulose, Chiralcel OJ; a modified cyclodextrin. Cyclobond I 2000 RSP, and a protein, Chiral-AGP. Retention factors, enantioselectivity, efficiency, and resolution were tested by modifying the composition of the mobile phase as well as the temperature. The mechanism for the chiral recognition of methadone on each support was discussed. Optimal chromatographic parameters were obtained for the three supports tested, and methadone enantiomers were separated in less than 20 minutes. The cellulose-based column gave the best resolution, but this CSP was not adapted to clinical analyses of methadone. Under optimized conditions, the cyclodextrin- and protein-based columns allowed an excellent separation of methadone enantiomers, but no interference with the primary metabolite was found only with Chiral-AGP.