Absolute rates of gluconeogenesis and of the citric acid cycle were assessed in livers isolated from 24-h starved rats, perfused with physiological concentrations of [3-13C]lactate and [3-13C]pyruvate +/- 0.2 mM octanoate. Calculations are based on (i) the 13C-labeling pattern of glutamate determined by gas chromatography-mass spectrometry combined with isotopomer analysis, (ii) substrate balance, and (iii) equations developed by Magnusson et al. (Magnusson, I., Schumann, W. C., Bartsch, G. E., Chandramouli, V., Kumaran, K., Wahren, J., and Landau, B. R. (1991) J. Biol. Chem. 266, 6975-6984) based on a citric acid cycle model proposed by Katz (Katz, J. (1985) Am. J. Physiol. 248, R391-R399). Glutamate, isolated from liver extracts, is enzymatically or chemically converted to gamma-aminobutyrate, alpha-hydroxyglutarate, isocitrate, and glutamine before mass spectrometric analysis. General equations have been developed (“Appendix I”) to determine the isotopic enrichment of each carbon of glutamate from the isotopic enrichment of fragments obtained from the mass spectra of trimethylsilyl or t-butyldimethylsilyl derivatives of glutamate and of derived compounds (“Appendix II”). In the presence of octanoate, (i) the rate of the citric acid cycle decreases from 0.25 to 0.13 mumol/min x g wet weight which are one-third and one-sixth of the rate of pyruvate carboxylation, and (ii) the rate of gluconeogenesis increases from 0.65 to 0.83 mumol/min x g wet weight. The rate of pyruvate carboxylation is 13 and 34-fold faster than that of pyruvate dehydrogenation in the absence or presence of octanoate, respectively. The rate of oxaloacetate to fumarate interconversion is at least six times greater than that of the citric acid cycle. Our data closely agree with those obtained by Magnusson et al. who used a non-invasive “chemical biopsy” of the human liver and support the use of labeled lactate and/or pyruvate for tracing hepatic metabolism in vivo.