A crude preparation of rat liver acyl-CoA synthetase [EC 6.2. 1.3] was stabilized by EDTA. It was found that acyl-CoA synthetase increased 2 to 3 fold in the livers of starved and diabetic rats, and of rats fed on a high fat diet. Among various intermediates of the tricarboxylic acid cycle, citrate and isocitrate inhibited acyl-CoA synthetase. A physiological significance of the citrate inhibition was discussed.