The nonspecific transaminase inhibitor aminooxyacetate (AOA) has multiple influences on the dynamics of 13C appearance in glutamate in rat hearts as measured by 13C nuclear magnetic resonance (NMR) without altering O2 consumption or tricarboxylic acid (TCA) cycle flux. These include the following: 1) a reduced rate of 13C enrichment at glutamate C3 and C4; 2) a near coalescence of the C3 and C4 fractional enrichment curves; 3) a dramatic alteration in the time-dependent evolution of the glutamate C4 multiplets, C4S and C4D34; and 4) a decrease in the NMR visibility of glutamate. A fit of the 13C fractional enrichment curves of glutamate C4 and C3 in the absence of inhibitor to a kinetic model of the TCA cycle gave values for transaminase flux of 7.5 mumol.min-1.g dry wt-1 and TCA cycle flux of 7.5 mumol.min-1.g dry wt-1, thereby confirming reports by others that the kinetics of 13C enrichment of glutamate C3 and C4 in heart tissue is significantly affected by flux through reactions other than TCA cycle. The 13C fractional enrichment data collected in the presence of 0.5 mM AOA could not be fitted using this same kinetic model. However, kinetic simulations demonstrated that the time-dependent changes in C4S and C4D34 are only consistent with a 10-fold reduction in the size of intermediate pools undergoing rapid turnover in the TCA cycle. We conclude that inhibition of glutamic-oxalacetic transaminase by AOA effectively reduces the size of the alpha-ketoglutarate pool in rapid exchange with the TCA cycle. Our data indicate that changes in glutamate multiplet areas in the 13C NMR spectra of heart (as demonstrated by glutamate C4S and C4D34) are more sensitive to alterations in metabolic pool sizes in exchange with the TCA cycle than are measurements of 13C fractional enrichment at glutamate C3 and C4.