The appearance of 13C label in glutamate has been used to quantify cellular tricarboxylic acid (TCA) cycle activity using 13C-NMR spectroscopy. Glutamate is linked to the TCA cycle by the amino-transferase reactions, however the consequences of alterations in amino-transferase activity on glutamate labelling kinetics, at a constant total tricarboxylic acid cycle activity, have not been investigated. Aspartate amino-transferase activity in [2-13C]acetate-perfused beating rat hearts was found to be similar to total TCA cycle flux in the presence of normal perfusion conditions and was reduced by more than 50% with the subsequent administration of amino-oxyacetic acid (AOA). AOA did not reduce contractile or kinetic measures of total TCA cycle flux, but did slow the 13C labelling of glutamate, in accord with current mathematical predictions. The impact of similar reductions in amino-transferase activity on estimates of total TCA cycle flux derived from several previously reported methods was also evaluated. Because total TCA cycle and the amino-transferase activities both affect the kinetics of 13C-glutamate labelling and because the amino-transferase activities are often unknown under physiologic conditions and can be reduced under pathologic conditions, the calculation of total TCA cycle flux from 13C-NMR data in the future is probably best accomplished either with a sufficiently sophisticated mathematical model that assesses amino-transferase activity or with an empiric model that is relatively insensitive to variations in amino-transferase activity.