1. 1.Malonyl-CoA decarboxylase (CE 4.1.1.9) was purified 51-fold from beef liver homogenate, by acid extraction, ammonium sulphate fractionation, precipitation at pH 5.8 and gel chromatography on Sephadex G-200. The molecular weight was estimated to be 250 000. Phosphate is an activator of the enzyme. The velocity measured at a hight substrate concentration (several times K m ) is constant between 40 and 200 mM phosphate. Within this range, the K m for malonyl-CoA decreases with increasing phosphate concentrations. The enzyme possesses optimal activity at pH 5 and 9, and a minimal activity between pH 6 and 7. 2. 2.Acetyl-CoA, propionyl-CoA and palmitoyl-CoA are non-competitive inhibitors with K i values of 0.66 mM, 0.28 mM and 2.3μM, respectively. The inhibitory action of palmitoyl-CoA decreases with increasing protein concentrations. The enzyme is partially competitively inhibited by aromatic aldehydes. 3. 3.The malonyl-CoA decarboxylase activity of intact mitochondria is latent, and this latency is not abolished by the addition of carnitine. Malonyl-CoA is not a substrate for carnitine acetyltransferase (EC 2.3.1.7). Since malonyl-CoA decarboxylase is located in the mitochondrial matrix space, it cannot affect the extra-mitochondrial malonyl-CoA.