Pre-steady-state13C nuclear magnetic resonance (NMR) spectra can provide a nondestructive probe of metabolic events associated with the physiology of intact organs. Therefore, the relation between phosphorylation state and intermediary metabolism in rabbit hearts, oxidizing [2-13C]acetate, was examined with a combination of31P and13C NMR. Multiple enrichment of the tissue glutamate pool with13C as an index of metabolic turnover within the tricarboxylic acid cycle was readily observed as a function of work load. Dynamic changes in pre-steady-state13C spectra evolved according to work load and correlated closely to respiratory rate in rabbit hearts perfused 1) under normal conditions (n=7), 2) at basal metabolic rates (20 mM KCI arrest,n=5), 3) and at heightened contractile state (10−7M isoproterenol,n= 7). The ratio of signal intensity arising from the secondary labeling sites within glutamate (C-2 and C-3) to that of the initial labeling site (C-4) reached steady state within 8.5 minutes in isoproterenol-treated hearts versus 18.5 minutes in control hearts. Work load did not affect glutamate concentration or fractional enrichment at the C-4 position, although an unlabeled fraction of glutamate persisted. Arrested hearts displayed slowed evolution of steady-state13C enrichment with increased contributions from anaplerotic sources for tricarboxylic acid intermediate formation (32%) as compared with control (9%). Thus, the response of mitochondrial dehydrogenase activity to the demands of cardiac performance is likely to influence the recruitment of anabolic sources supplying the tricarboxylic acid cycle. In contrast to changes in13C NMR spectra, relative high-energy phosphate levels from31P spectra were similar at all work loads, with no apparent differences in free ADP, as indicated by similar phosphocreatine to ATP ratios (2.1–2.3). These findings suggest that regulation of respiratory activity is mediated by changes in the rate of reducing equivalent generation from substrate oxidation. (Circulation Research1992;70:576–582)