High-performance frontal analysis (HPFA) wasapplied to investigate the binding properties of semotiadil(R-isomer, Ca-channel blocker) and levosemotiadil (S-isomer,Ca- and Na-channel blocker), an enantiomeric pair of drugs under devel-opment, to human serum albumin (HSA). An on-line HPLC systemconsisting of a HPFA column, an extraction column and an analytical HPLC column was used to determine the unbound concentrations of theseenantiomers. The experimental data were subjected to Scatchard analyses to estimate the binding parameters. The binding affinity of levosemo-tiadil(K=6.59×10⁵ M^-1, n=0.97) is aboutthree-times stronger than that of semotiadil (K=2.15×10⁵M^-1, n=0.99). These results did not change in thepresence of warfarin, but were significantly decreased by the addition ofdiazepam, indicating that both enantiomers are bound at the diazepam siteon HSA molecule.