To what extent does glutamine turnover keep pace with oxidative metabolism in the rat heart? To address this question, the following groups of substrates were presented to the isolated, working rat heart: (1) glucose (5 mM), insulin (40 µU/ml) and [2-¹³C]acetate (5mM) (high workload, n= 5); (2) pyruvate (2.5 mM) and [2-¹³C]acetate (5 mM) (normal workload, n= 5); or (3) propionate(1 mM) and [2-¹³C]acetate (2.5mM) (normal workload, n=3). In a subset of these experiments the exchange of glutamate and glutamine was quantified by separation with ion exchange chromatography and analysis by gas chromatography-mass spectrometry. There was an apparent equilibration of mass isotopomers of glutamate and glutamine after 50 minutes of perfusion, although the extent of equilibration was not determined. The fractional enrichment in glutamine was 31% of the enrichment of glutamate with the three different perfusates. From high resolution NMR spectra, we found a ratio of glutamine to glutamate content of 94.1%, 53.4% and 96.9%, respectively, for each experimental group. In experiments for which [1- ¹³C]L-glutamine (5mM) was included in the perfusate of group 2 (above), [1-¹³C]glutamine was detected in the heart, but transfer of ¹³C from glutamine to glutamate was not detected (n=4). We conclude that in the perfused working heart, production of glutamine by amidation of glutamate takes place and can be detected, whereas the reverse process, generation of glutamate from glutamine, remains undetected.