1. 1. Pyruvate and a number of other α-keto acids have been used as substrates for the acylation of carnitine by intact mammalian mitochondria. Acetyl- and/or propionylcarnitine were the only detectable reaction products, irrespective of the chain length of the α-keto acid used. 2. 2. These results indicate that a β-oxidation takes place prior to a transacetylation to carnitine. 3. 3. The yields of acylcarnitines decreased with increasing chain length of the α-keto acid. 4. 4. When α-ketovalerate was the substrate a stimulation of the citric acid cycle with succinate inhibited the acetylation of carnitine, whereas an inhibition of the citric acid cycle with malonate stimulated the acetylation. 5. 5. Acetyl-CoA was shown to be the acetyl donor in the mitochondrial acetylation of carnitine. 6. 6. The hypothesis that acetylcarnitine acts as a carrier of activated acetyl groups through the mitochondrial membrane is discussed. 7. 7. The possible use of the mitochondrial carnitine acetyltransferase (acetyl-CoA: carnitine O-acetyltransferase, EC 2.3.1.7) in a quantitative enzymatic assay procedure for l-carnitine is suggested.