Succinyl-CoA inhibits 3-hydroxy-3-methyl-glutaryl-CoA lyase (EC 4.1.3.4) when added to purified preparations of the enzyme. 2. The apparent Ki value is 2.1 x 10(-4) mol/l and the inhibition has the features of a partially competitive inhibition. 3. The effect of succinyl-CoA both added and enzymically produced on the lyase activity of sonically disrupted rat liver mitochondria results in decreased acetoacetate formation. 4. This occurs with mitochondria obtained from normal, starved and streptozotocin-diabetic rats.