Carnitine is an essential component in tissues of animals, higher plants and many microorganisms. Whereas the L-carnitine enantiomer plays an important role in the metabolism of long chain fatty acids, D-carnitine has a considerable toxic influence on biochemical processes. The analytical separation of D-and L-carnitine depends upon derivatization with UV-or fluorescently active substances, e.g. FMOC and (+)/(−)-FLEC. The separation of diastereomeric (+)- and (−)-FLEC carnitine esters was performed successfully with capillary zone electrophoresis (CZE) and HPLC, after optimization of the derivatization process and of the composition and pH of the buffer, using UV- and fluorescence detection. With HPLC separation a detection limit of the carnitine esters of 5 μmol/l when using fluorescence detection was achieved. With both separation systems baseline resolution and short analysis times could be obtained. The enantiomeric FMOC derivatives could be separated using the electrophoretic system and acidic buffers with high concentrations of an osmotic flow modifier together with γ-cyclodextrine as chiral selector. The applicability of the optimized separation conditions are demonstrated in the analysis of agar culture medium inoculated withPseudomonas putidaand of pharmaceutical formulations. In all samples very low amounts of D- or L-carnitine could be determined in the presence of the other enantiomeric form. Problems caused by the impurity of the carnitine standards or the derivatization agent (+)/(−)-FLEC are di