A combined isotopic steady state and in vivo isotopic non-steady state analysis was used to calculate tricarboxylic acid cycle flux in livers of anesthetized rats infused with ethanol. In vivo 13C NMR spectroscopy was used to non-invasively observe label turnover of [4-13C]glutamate, [4-13C]glutamine, and [2-13C]glutamate/glutamine in liver following a bolus intravenous infusion of [2-13C]ethanol. The isotopic steady state analysis of [2-13C], [3-13C], and [4-13C]glutamate isotopomers (Malloy, C. R., Sherry, A. D., and Jeffrey, F. M. H. (1988) J. Biol. Chem. 263, 6964-6971) in liver extracts was used to indirectly calculate the anaplerotic flux (0.90 +/- 0.07 x citrate synthase flux) and [2-13C]acetyl-CoA fractional enrichment (51.4 +/- 3.4%). The [4-13C]glutamate, [4-13C]glutamine, and [2-13C]glutamate fractional enrichments determined in liver extracts were 23.0 +/- 1.1, 17.2 +/- 1.5, and 7.7 +/- 0.5%, respectively. These data in addition to blood [2-13C]acetate and [4-13C]glutamine enrichment time course data were used in conjunction with a metabolic steady state mathematical analysis designed to account for liver glutamate and glutamine label dilution as a consequence of glutamine exchange with blood to calculate the tricarboxylic acid (tca) cycle flux (Vtca = 0.33 +/- 0.09 micromol/g wet weight/min) in liver. In summary, It is possible to detect 13C labeling of glutamate and glutamine in liver via non-invasive 13C NMR. Additionally, the in vivo 13C labeling kinetics of glutamate and glutamine in liver and glutamine in blood may be used to calculate the liver tricarboxylic acid cycle flux.