Several studies using exogenous pyruvate as substrate, have suggested that there are separate intracellular pyruvate pools in cardiac cells. Such heterogeneity in intracellular pyruvate has important implications for our understanding of both oxidative and nonoxidative glucose metabolism. Because pyruvate is not a major substrate for the heart in vivo, we wished to determine if there was pyruvate compartmentation with glucose as substrate. Hearts were isolated from male Sprague-Dawley rats and retrogradely perfused (Langendorff) with a modified Krebs-Henseleit buffer containing [1- 13C]glucose for 5-115 min. At the end of each experiment, hearts were freeze-clamped and extracted for determination of the fractional enrichment of alanine, lactate, and acetyl CoA using 1H- and 13C-nuclear magnetic resonance spectroscopy. The fractional enrichment of alanine at the C-3 position was significantly higher than that for lactate at 55 min [43.6 ± 2.8 vs. 27.2 ± 4.6% (SD), n = 5, P < 0.003]. The differences in steady- state enrichment between lactate and alanine were not due to differences in the rate of labeling of these metabolites. The mean steady-state lactate enrichment was higher in the perfusate samples compared with the tissue samples from the same experiments (46.6 ± 2.2 vs. 30.5 ± 2.5%, n = 3, P < 0.005). Because fractional enrichment of alanine, acetyl CoA, and perfusate lactate are similar, we suggest that there is a separate nonexchanging pool of lactate rather than cytosolic compartmentation of pyruvate that has been previously proposed.