Carnitine acetyltransferase was purified from the citric acid producing A. niger mycelium with a protein band showing a relative molecular weight of 77,000 and a pH optimum of 7.3. The Km values for the purified enzyme for acetyl-CoA and for carnitine were 0.1 mM and 1 mM, respectively. Carnitine acetyltransferase was located both in the mitochondria and in the cytosol. Both mitochondrial and cytosolic enzyme were purified using ammonium sulfate precipitation, Mono Q and Superose 12 separation. Regarding the localization, except for maximum velocity, there were no differences observed in substrate specificity and inhibition. Inhibition of the enzyme with micromolar concentrations of Cu2+ could contribute to a greater citric acid biosynthesis. Carnitine acetyltransferase can be considered as an enzyme necessary for the transport of acetyl groups through mitochondrial membrane in both directions.