The mitochondrial membrane is not permeable to long-chain fatty acids; a multi-step process is therefore required for these compounds to be used by mitochondria. In the muscle cytoplasm, long-chain fatty acids are first activated by long-chain acyl-CoA synthetase (ACS) to their CoA thioesters. The CoA thioesters are subsequently linked with carnitine by the enzyme carnitine palmitoyltransferase I (CPT I) located on the inner side of the outer mitochondrial membrane. The acylcarnitine form of the long-chain fatty acid, palmitoylcarnitine, is then transferred across the inner mitochondrial membrane by carnitine:acylcarnitine translocase. Once in the mitochondrial matrix, it is converted back to free acyl-CoA derivative and carnitine by carnitine palmitoyltransferase II (CPT II) on the inner side of the inner mitochondrial membrane. Once carnitine is released, the long-chain acyl-CoA derivative enters the beta-oxidation pathway. With every complete cycle, a two-carbon fragment is cleaved and an acetyl-CoA molecule is released.