1. 3‐Hydroxy‐3‐methylglutaryl‐CoA (HMG‐CoA) synthase (EC 4.1.3.5) in extracts of rat liver mitochondria can be inactivated by succinyl‐CoA and activated by incubation in a medium designed to cause desuccinylation (‘desuccinylation medium’). 2. The enzyme is less active in extracts of whole liver from control rats than from rats treated with glucagon or mannoheptulose. Incubation in desuccinylation medium raises the activity in extracts from control rats to the same value as treated rats, suggesting that the extent of succinylation in vivo is greater in controls than in hormone‐treated animals. 3. This result is also obtained in liver homogenates and in isolated mitochondria. 4. Increasing the succinyl‐CoA content of mitochondria to the same high level lowers the enzyme activity to the same value in mitochondria isolated from control or treated rats. In each case subsequent incubation of the lysates in desuccinylation medium raises the enzyme activity by the same extent. 5. Measurement of the incorporation of radiolabel from 2‐oxo[5‐¹⁴C]glutarate into protein is consistent with the proposal that all these changes in activity in isolated mitochondria may be explained by changes in the extent of succinylation of the enzyme. 6. From these data and our earlier work we conclude that, in vivo, mitochondrial HMG‐CoA synthase in fed rats is normally substantially succinylated (about 40%) and inactivated, and that glucagon increases the activity of HMG‐CoA synthase by lowering the concentration of succinyl‐CoA and thus decreasing the extent of succinylation of the enzyme (to less than 10%). This may be an important control mechanism in ketogenesis.