Summary 1. In a reaction mixture containing KIC. Mg—, phosphate buffer and ATP, net acetoacetate formation from n-valerate, and n-caprylate by rat liver mitochondria proceeded readily. Under conditions of our experiments, metabolism of isovalerate, 3-methyl-2-butenoate, and 3-methyl-3 butenoate proceeded only in the additional presence of bicarbonate. Malonate markedly depressed metabolism of the latter three acids which occurred in the presence of bicarbonate. Cooxidation of citrate, glutamate, or a-keto-glutarate overcame this malonate inhibition. Malonate did not decrease the yield of acetoacetate from n-valerate, a-methyl-butyrate, or n-caprylate. 2. Mitochondria from both biotin-injected rats and biotin-deficient rats produced acetoacetate from n-valerate, a-methyl-butyrate and n-caprylate. The β-methyl fatty acids, isovalerate, 3-methyl-2-butenoate. and 3-methyl-3-butenoate were metabolized by mitochondria of biotin-injected rats but not by mitochondria of deficient rats.