The assay of oxaloacetate and (\alpha)-ketoglutarate in biological samples is complicated by their chemical instability and low concentrations. We present a quantitative assay for physiological concentrations of these metabolites by isotope dilution gas chromatographymass spectrometry. Samples are spiked with the corresponding internal standards of (\left[\mathrm{U}^{-13} \mathrm{C}{4}\right]) oxaloacetate and (\left[\mathrm{U}^{13} \mathrm{C}{5}\right] \alpha)-ketoglutarate prior to their treatment with hydroxylamine. After ethyl acetate extraction and evaporation of the organic phases, the oximes are converted to (t)-butyldimethylsilyl ethers and analyzed by selected ion monitoring gas chromatography-mass spectrometry of the ([\mathrm{M}-57]^{+})ion in electron impact. Although the internal standards of (\left[\mathrm{U}{-}{ }^{13} \mathrm{C}{4}\right]) oxaloacetate and (\left[\mathrm{U}{-}{ }^{13} \mathrm{C}{5}\right] \alpha)-ketoglutarate are not commercially available, they can easily be synthesized in (30 \mathrm{~min}) by reacting (\left[1,2,3,6-{ }^{13} \mathrm{C}{4}\right]) citrate with citrate lyase, and (L) (\left[\mathrm{U}{-1}{ }^{13} \mathrm{C}{5}\right] g l u t a m a t e) with pyruvate and glutamate-pyruvate transaminase, respectively. Because of their chemical instability, the internal standards are prepared on the day of the analysis. A stock solution of (\left[1,2,3,6-{ }^{13} C{4}\right]) citrate is prepared from (L-\left[U_{-}{ }^{13} C_{4}\right]) aspartate using citrate synthase and glutamate-oxaloacetate transaminase and then purified and kept frozen until required. The detection limit of the method is 0.05 nmol in a given sample. The method was applied to measurements of oxaloacetate and (\alpha)-ketoglutarate in human blood and rat liver. C 1995 Academic Press, Inc.