BACKGROUND: There is much interest in the recognition and determination the two ofloxacin enantiomers, not only from the point of view of investigating the pharmacokinetics of the enantiomers in vitro but also in the design and development of new chiral pharmaceutics. RESULTS: Chiral separation was performed on a C-18 column, in which the mobile phase consisted of a methanol-water solution (containing different concentrations of L-phenylalanine and copper sulfate) and its flow rate was set at 0.7 mL min(-1). The effect of different kinds and concentration of ligands, bivalent copper ion, organic modifier, ionic liquid modifier, pH of mobile phase, and temperature on enantioseparation were evaluated and the results show that the enantioselectivity was strongly affected by the pH and ligand concentration of the mobile phase. Under optimal conditions, baseline separation of the two enantiomers was obtained with a resolution of 4.69 in less than 40 min. CONCLUSION: The mechanism of chiral discrimination is based on the stabilities of the copper(II) binary complexes and their ternary diastereomeric complexes with amino acids formed in solution and stationary phase. The proposed method could be used for the quality evaluation of ofloxacin enantiomers. (C) 2009 Society of Chemical Industry