A new chromatographic procedure was developed for the separation of atenolol (ATN) enantiomers based upon chiral ligand-exchange principal. The separation was carried out on a C-8 column. L-alanine and Cu2+ were applied as chiral selector and central bivalent complexing ion, respectively. It was found that the kind of copper salt had vital effect on the enantioseparation. The separation on the C-8 stationary phase was more efficient than that on the C-18 column. The pH of mobile phase, organic modifier content of mobile phase, mole ratio of chiral ligand to bivalent ion and Cu(L-alanine)(2) concentration in the mobile phase were found to be important in enantiomers resolution efficiency. Water/methanol (70:30) mixture containing L-alanine-Cu2+ (2:1) was found to be the best mobile phase condition for ATN enantioseparation. The concentration of Cu(L-alanine)(2) complex in the mobile phase influenced either enantiomers resolution efficiency or the detection sensitivity. All effective parameters were optimized in order to satisfy both detection sensitivity of the method and its separation efficiency. The optimized HPLC method was utilized in some synthetic and human blood plasma samples. (C) 2013 Elsevier B.V. All rights reserved.