The influence of metabolic substrate on contractile strength, myocardial oxygen consumption (MVO2), high- and low-energy phosphate levels, and intracellular pH were determined in isovolumically contracting isolated rat hearts perfused with solutions containing either glucose or hexanoate at both high and low coronary perfusion pressures (CPP). Contractile strength was not significantly influenced by substrate at a CPP of 80 mmHg. As coronary flow was decreased, developed pressure measured at a fixed left ventricular volume (LVV) was lower during hexanoate than glucose perfusion. The relationship between MVO2 and mechanical work determined at a CPP of 80 mmHg over a range of LVVs was shifted upward in a parallel manner when substrate was switched from glucose to hexanoate. The MVO2-work relationship measured at a fixed LVV but over a range of coronary flows (7-20 ml/min) was also parallel shifted upward on switching from glucose to hexanoate. Basal MVO2 was greater during hexanoate than glucose perfusion by an amount that accounted for two-thirds the total increase in MVO2 observed between the substrates under unloaded beating conditions. The remainder of the difference was attributed to increased energy requirements for excitation-contraction coupling. Inorganic phosphate concentrations increased more and phosphocreatine concentrations decreased more during low-flow conditions (3 ml/min) when hearts were perfused with hexanoate compared with glucose. Thus hexanoate decreases myocardial efficiency compared with glucose in large part by increasing non-work-related oxygen demands. This inefficiency impacts adversely on contractile strength and high-energy phosphate concentrations at low coronary flows.