Abstract: Resolution of enantiomers of lomefloxacin by HPLC was proposed using the Chiralcel OJ column as stationary phase with column temperature of 30 ℃, and a mixture of n-hexane and iso-butanol (90+10) as mobile phase with a flow-rate of 0.6 mL·min-1. UV-detection at the wavelength of 287 nm was adopted in the determination. Based on the solute stoichiometric displacement model of retention and the thermodynamic theory, mechanism of resolution of enantiomers of lomefloxacin on this chiral column was studied. It was found that values of lgI and Z of the enantiomer with shorter retention time were smaller than that of the enantiomer with longer retention time. Good linearity of the Van′t Hoff curve was obtained when plotting lnα vs. 1/T, within the testing temperature range; and the difference between enthalpy change and entropy change was shown to be a negative value. It was thus illustrated that the chiral resolution of the enantiomers was an enthalpy-controlled process.