In the isolated rat heart perfused with glucose as substrate, measurements were made of perfusate and tissue lactate and pyruvate concentrations, and of tissue α-glycerophosphate and dihydroxyacetone phosphate concentrations. The conditions studied included increased heart work, anoxia, the addition of insulin, acute alloxan diabetes, chronic streptozotocin diabetes, and the addition of ketone bodies. True intracellular lactate values (estimated from the lactate and sorbitol spaces) and apparent tissue values exceeded perfusate values, showing the existence of a lactate concentration gradient. During anoxia, the lactate gradient diminished. Cytoplasmic free NAD+/NADH ratios calculated from the extracellular lactate/pyruvate ratio were similar to and changed in the same direction as the NAD+/NADH ratios calculated from the tissue α-glycerophosphate/dihydroxyacetone phosphate ratios in non-diabetic hearts. In diabetio hearts, extracellular and tissue lactate/pyruvate ratios gave different values for the cytoplasmic free NAD+/NADH ratios than did tissue α-glycero-phoaphate/dihydroxyacetone phosphate ratios. These discrepancies were associated with accumulation of pyruvate in the perfusate and the tissue. Changes in the extracellular lactate/pyruvate ratio gave a better differentiation between acute heart work and acute anoxia than did changes in the heart lactate/pyruvate ratio. The use of extracellular lactate/ pyruvate measurements in assessing the state of myocardial oxygenation is supported by these observations, provided that nutritional factors are taken into account and the diabetic state is excluded.