The effects of various substrates on the concentrations of free amino acids, citric acid cycle intermediates and acylcarnitines were studies in perfused hindquarter of rat in presence of glucose and insulin in order to assess regulatory mechanisms of the level of citric acid cycle intermediates in skeletal muscle. 1. Acetate and acetoacetate effected a significant increase in the level of citrate cycle intermediates and accumulation of acetylcarnitine. These changes were accompanied by a reduction in the level of alanine. The concentration of AMP was significantly elevated. 2. Muscle mitochondria fixed 14CO2 in the presence of pyruvate. The products were identified as malate or citrate when whole and disintegrated mitochondria were used respectively. The fixation was greatly stimulated by acetylcarnitine. 3. Acetylcarnitine inhibited the production of pyruvate from malate by muscle mitochondria. 4. Perfusion with 2-oxoisocaproate and 2-oxoisovalerate promoted increases in the level of citric cycle intermediates, a drop in both alanine and glutamate, and accumulation of branched-chain acylcarnitines. 2-Oxoisocaproate also caused a reduction of alanine released from the muscle. 5. Perfusion with leucine and valine did not change the concentration of citric acid cycle intermediates, but elevated glutamate and still more the concentration of alanine. 6. It is concluded that citric cycle intermediate level in the perfused resting muscle is modified by a) conditions which change the concentration of acetyl-CoA and thereby modify the rate of pyruvate carboxylation and decarboxylation of malate via malic enzyme b) conditions which change the concentration of pyruvate cause changes in alanine and cycle intermediates in the same direction via transamination reactions c) conditions which change the concentrations of 2-oxoacids which are converted to cycle intermediates via oxidation.