AbstractThe enantioselective and diastereoselective resolutions of the stereoisomers of Nα‐aspartyl‐phenylalanine 1‐methyl ester (APME) have been accomplished on an HPLC chiral stationary phase based upon α‐chymotrypsin (the ACHT‐CSP) with observed enantioselectivities (α1) for the DL‐/LD‐enantiomers of as high as 29.17 and for the DD‐/LL‐enantiomers of as high as 28.97. In addition, the effect on the chromatographic retention of the APME stereoisomers of the activity of the ACHT and the composition of the mobile phase—structure of the anionic component, molarity, and pH—have been studied. The results of this study suggest that the aspartyl moiety and/or the aspartyl‐phenylalanine amide linkage play key roles in the observed enantioselectivity; the APME stereoisomers containing L‐phenylalanine, i.e., DL‐ and LL‐APME, bind at a different site in the ACHT molecule (the L‐Phe site) than the APME stereoisomers containing D‐phenylalanine (the D‐Phe site); and the observed enantioselectivity is a measure of the difference in the binding affinities at the two sites rather than the consequence of di