A MEKC methodology with UV detection was developed for the enantioselective separation of selenomethionine (SeMet). The use of (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC) as chiral derivatization reagent to form SeMet diastereomers enabled their subsequent separation using ammonium perfluorooctanoate (APFO) as a volatile pseudostationary phase. The effect of APFO concentration and pH, temperature, injection volume, and derivatization conditions (time and FLEC/SeMet ratio) were evaluated in order to select the best separation conditions. A chiral resolution of 4.4 for DL-SeMet was achieved in less than 6 min using 100 mM APFO at pH 8.5 as electrophoretic buffer. Satisfactory results were obtained in terms of linearity, precision (RSD from 3.4 to 5.1% for migration times and from 1.8 to 4.6% for corrected peak areas), accuracy, and LODs (3.1 x 10(-6) M and 3.7 x 10(-6) M for d and l enantiomers, respectively). The method was successfully applied to the determination of l-SeMet in food supplements.