On perfusing the isolated rat heart with substrate-free medium myocardial contractility was maintained for 30 min and thereafter declined towards zero in a subsequent period of 90 min. A slight decrease in heart rate was also apparent. In hearts deprived of the exogenous substrate, a marked reduction in the levels of glycogen, various glycolytic intermediates, triglycerides, high energy phosphate stores, and amino acids, which are most easily transaminated to Krebs cycle intermediates, was observed. Fluorescence due to reduced pyridine nucleotides, recorded from the surface of the heart, decreased during perfusion with substrate-free medium. Chemical analysis of substrate-depleted hearts also revealed a decrease in the concentrations of both NADH and NADPH. Perfusion of the heart with medium containing glucose for 2 h maintained myocardial contractility and slightly increased glycogen levels without appreciably affecting the slight decline in heart rate seen after perfusion with substrate-free medium. The presence of glucose in the perfusion medium partially prevented the changes in the levels of high energy phosphate stores, glycolytic intermediates, amino acids, triglycerides, and reduced pyridine nucleotides. Damage to the ultrastructure and swelling of mitochondria as seen on perfusing the hearts with substrate-free medium were also prevented partially by the presence of glucose in the perfusion medium. The ability of exogenous glucose to restore the contractile force and high energy phosphate stores towards the normal level declined over a 2 h period of preperfusion of the heart with substrate-free medium. The data support the general role of exogenous glucose in the maintainance of myocardial contractility fully and of metabolic and ultrastructure of the heart partially.