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Positron emission tomography is a unique noninvasive imaging technique that provides cross-sectional images of radiotracer concentrations in myocardium and permits measurement of blood flow as well as metabolism. Ammonia and glutamate have been labeled with the positron-emitter 13N (half-life 10 minutes) for use with positron emission tomography as tracers of flow and metabolism, respectively. In order to characterize the fate of these 13N-labelled compounds in myocardium, isolated rabbit interventricular septa were used to study the kinetics of [13N] glutamate ([13N]glu) and 13NH3 under aerobic and anoxic conditions. Tissue analyses 6 minutes after injection of a [13N]glu bolus into myocardium revealed that 70% of the 13N-label was present in [13N]glu 12%, 11%, and 4% in [13N]alanine ([13N]ala), [13N]aspartate ([13N]asp), and [13N]glutamine ([13N]gln), respectively. The corresponding relative specific activities were 1.0:0.4:0.5:0.01. Anoxia resulted in a significant increase in [13N]ala with a reduction in [13N]glu. This was consistent with increased pyruvate production due to increased anaerobic glycolysis and transamination of pyruvate with [13N]glu to yield [13N]ala. In support of this, addition of 2 mM pyruvate to the perfusate under control conditions produced a tissue distribution of 13N similar to that with anoxia. Six minutes after a bolus of 13NH3 during both control and anoxic conditions, 60% of the tissue 13N-label was in [13N]gln with no detectable amounts in other amino acids. The rest of the 13N-label was in 13NH3. Time-activity curve analyses demonstrated that anoxia significantly reduced the tissue retention of 13N-label from 13NH3 but not from [13N]glu. Thus, 13N from 13NH3 and [13N]glu was retained in tissue by different mechanisms involving glutamate, which were affected differentially by anoxia. These results suggest that positron emission tomography imaging with 13NH3 and [13N]glu in combination may be useful in identifying ischemic myocardium.