We investigated the hypothesis that one mechanism underlying fatty acid toxicity is the selective inhibition of rate-limiting and/or regulated tricarboxylic acid cycle and related enzymes by fatty acyl coenzyme A (CoA) derivatives by examining the effects of several fatty acyl CoAs on purified citrate synthase (CS) and glutamate dehydrogenase (GDH). The results indicate that, at pathophysiological levels, palmitoyl CoA, a long-chain acyl CoA, is a potent inhibitor of CS and GDH with IC50 values of 3-15 microM. At much higher levels (in the pathological and toxicological range), octanoyl and decanoyl CoA (medium-chain acyl CoAs) inhibited both enzymes with IC50 values of 0.4-1.6 mM. Butyryl CoA, a short-chain acyl CoA, inhibited CS (IC50 = 0.9 mM) at toxicological levels but inhibited GDH poorly. These results suggest that the long-chain fatty acyl CoA inhibition of CS and GDH may assume some pathophysiological importance in fatty acid toxicity and in metabolic encephalopathies in which organic acidemia is persistent. The findings also provide additional support for the original hypothesis.