Earlier studies with the perfused rat heart have shown that the rate of glycolysis was inhibited by the addition of fatt) y acids or ketone bodies to the perfusion medium (l-4). The rate of glucose phosphorylation was shown to be an important factor determining the over-all rate of glucose uptake by the heart when membrane transport was stimulated by insulin. On the basis of changes in the tissue content of glucose B-phosphate, fructose 6-phosphate, and fructose 1, 6-diphosphate in the presence and absence of fatty acids, ketone bodies, or pyruvate, Newsholme, Randle, and Manchest’er (2) proposed that phosphofructokinase was the rate-limiting step of glycolysis in the perfused rat heart, and t, hat the activit, y of phosphofructokinase was decreased during the oxidation of these metabolic substrates. Decreased phosphofructokinase activity resulted in increased levels of glucose-6-P, and it was further proposed that hexokinase, being product-inhibited, was limited by the accumulation of glucose-6-P. Reports from several laboratories (5-8) have since suggested that citrat, e may have an important role in the regulation of glycolysis, as indicated by the ability of citrate to inhibit phosphofructokinase in the cell-free system, and its accumulat’ion in cardiac muscle under conditions of low carbohydrate utilization (6-8). Variations in the citrate content of a tissue such as the heart, which maintains a fairly constant respiratory ac-Gvity under conditions of constant work, is of great interest in terms of the over-all regulation of t, he citric acid cycle, and is a problem which merits furt’her attention. In this investigation, isotopic glucose was used to determine the major pathways of glucose metabolism in the presence and absence of insulin: after the addition of either acetate or pyruvate to the perfusion medium. These substrates were chosen as suitable fuels to compete with glucose for respiration, since they have previously been shown to be readily metabolized by the perfused rat heart (9, 10). The levels of most of the glycolytic intermediates, the adenine and pyridine nucleotides, and several of the citric acid cycle intermediates and related amino acids have been measured after the addition of either acetate or pyruvate to hearts perfused with glucose, in order to gain further insight into the mechanisms involved in the control of glycolytic activity. The mass action ratios of the glycolytic reactions, calculated on the basis of the observed intermediate levels,