The flux of glucose entering the glycolytic pathway under various metabolic conditions has been indirectly monitored in the Langendorff perfused rat heart using 31P-NMR spectroscopy. By totally inhibiting (>95%) glyceraldehyde-3-phos-phate dehydrogenase with low concentrations of iodoacetic acid (0.2 mM) in the perfusion medium, active glycolysis results in the accumulation of sugar phosphate species (fructose 1,6-bisphosphate, dihydroxyacetone phosphate, and glyceraldehyde 3-phosphate) which can be observed in the 31P-NMR spectrum. Using this technique, it has been shown that butyrate (10 mM) in the perfusion medium decreases the flux through the initial steps of the glycolytic pathway by at least 6-fold and that both glucose phosphorylation and glycogenolysis are inhibited. Upon total global ischemia in the presence of both glucose and butyrate, the glycolysis rate is stimulated approx. 100-fold. © 1988.