We determined the optical isomer of the 2-hydroxyglutaric acid (2HG) that was elevated in the urine of five Japanese children with a mild form of glutaric aciduria type II (GA2), caused by a deficiency of electron transfer flavoprotein (ETF) or ETF-ubiquinone oxidoreductase (ETF-QO). The D- and L-enantiomers of 2HG were separated by capillary gas chromatography with a combination of (S)-(+)-2-octanol derivatization and chromatography on a DB-1 column. The isomer that was elevated in GA2 patients was predominantly the D-enantiomer, an observation that may serve as an additional marker for the biochemical diagnosis of GA2. D-2HG dehydrogenation, but not L-2HG dehydrogenation is apparently blocked in GA2. A specific D-2HG dehydrogenase or D-2HG-CoA dehydrogenase may be metabolically linked to ETF and ETF-QO in the mitochondria.