SummaryThe absolute separation of the four stereoisomeric configurations of methylcitric acid can be achieved on a nonchiral stationary phase SE30 capillary column using the correspondingO‐acetylated (tri‐(−)‐2‐butyl ester derivatives. Identification of the separated isomers was done using methylcitric acid produced bysi‐citrate synthase and methylcitrate synthase ofCandida lipolitica. si‐Citrate synthase produces the (2S,3S)‐, (2S,3R)‐ and a small amount of the (2R,3S)‐isomers. Methylcitrate synthase produces the (2R,3S)‐isomer, indicating that this enzyme is more stereospecific than the animal citrate synthase enzyme. The (2R,3R)‐isomer may act as an inhibitor of aconitase.