The transport of metabolites across the mitochondrial membrane is regulated by specific exchange and shuttle systems that are often dependent on the mitochondrial membrane potential. Thus, metabolite concentrations in the cytosol and mitochondrial compartments are largely determined by the energy state of the cardiac muscle cell. The purpose of this study was to investigate metabolic compartmentalization in ventricular myocytes isolated from newborn (< 24 h) swine hearts. Furthermore, the effect of respiratory inhibition on these distribution patterns was examined. Freshly isolated cells contained 33 nmol of ATP and 37 nmol of total adenine nucleotides (AN) per mg of myocyte protein. Rapid digitonin fractionation indicated that 95% of ATP and 86% of AN were cytosolic, whereas > 50% of the pyridine nucleotides were mitochondrial. With 11 mM added glucose, myocytes treated with the respiratory inhibitor, rotenone, maintained ATP at 88% of that of aerobic myocytes, but phosphocreatine declined by 50% over 30 min. Rotenone treatment caused the mitochondrial NAD/NADH ratio to decline from 1.2 to 0.06, whereas the cytosolic pyridine nucleotides remained > 90% oxidized. Total adenine and pyridine nucleotide content and their compartmentalization were unaffected by respiratory inhibition. Comparisons of metabolite content and respiratory activity between isolated piglet mitochondria and the mitochondrial compartment of piglet myocytes indicated that mitochondria account for approximately 30% of total myocyte protein. A similar value (29%) was obtained for the aqueous volume fraction of the in situ mitochondrial matrix using the 4000 Mr 14C-labeled polyethylene glycol-impermeable 3H2O spaces of intact and lysed myocytes. These results are comparable to literature values for myocardium from other species and age groups.