Heart and liver mitochondrial, as well as liver peroxisomal, carnitine acetyltransferase was purified to apparent homogeneity and some properties, primarily of heart mitochondrial carnitine acetyltransferase, were determined. Hill coefficients for propionyl-CoA are 1.0 for each of the enzymes. The molecular weight of heart mitochondrial carnitine acetyltransferase, determined by SDS-PAGE, is 62,000. It is monomeric in the presence of catalytic amounts of substrate. Polyclonal antibodies against purified rat liver peroxisomal carnitine acetyltransferase precipitate liver and heart mitochondrial and liver peroxisomal carnitine acetyltransferase, but not liver peroxisomal carnitine octanoyltransferase. Liver peroxisomes, mitochondria, and microsomes and heart mitochondria all give multiple bands on Western blotting with the antibody against carnitine acetyltransferase. Major protein bands occur at the molecular weight of carnitine acetyltransferase and at 33 to 35 kDa.