Mitochondria of bovine liver contain acyl CoA synthetases necessary for the uptake of propionate, butyrate, and valerate whereas acetate is bound only weakly. Purification of these enzymes separated a distinct propionyl CoA synthetase highly specific for propionate and acrylate and a butyrate-activating fraction with broad substrate specificity for short and medium chain fatty acids. Evidence from kinetic studies and sucrose density centrifugation suggested that this latter fraction was composed of two enzymes, a butyryl CoA synthetase and a valeryl CoA synthetase. The apparent molecular weights of the propionyl, butyryl, and valeryl CoA synthetases were 72,000, 67,000, and 65,000. The Michaelis-Menten constants of propionyl CoA synthetase for propionate, adenosine 5'-triphosphate, and coenzyme A were 1.3 x 10(-3)M, 1.3 x 10(-3)M, and 6.3 x 10(-4)M. Enzyme activity is regulated by the concentration of propionate in portal blood. Relative to propionyl, butyryl, or valeryl CoA synthetases little acetyl CoA synthetase could be demonstrated. In ruminants hepatic metabolism is such that use of acetate as an energy source is minimum. This ensures that an alternative energy source to glucose, as acetate units, will reach the extrahepatic tissues. Separation of a distinct propionyl CoA synthetase regulated by the concentration of propionate in portal blood is significant because a primary role of ruminant liver is to synthesize glucose from ruminally derived propionate.