Certain amino acids and other compounds are metabolized via: Propionyl-CoA carboxylase in equilibrium D-methylmalonyl-CoA racemase in equilibrium L-methylmalonyl-CoA (adenosylcobalamin) mutase in equilibrium succinyl-CoA in equilibrium tricarboxylic acid cycle. In cobalamin deficiency and in genetic disorders involving adenosylcobalamin or the mutase, large amounts of methylmalonic acid are excreted in the urine. Its origin is unknown, however, since nonesterified methylmalonic acid is not present in the above or other known pathways. To investigate the origin of methylmalonic acid, we fractionated rat liver by gel filtration and found a single peak (Mr = 35,000) of activity for the hydrolysis of DL-methylmalonyl-CoA to methylmalonic acid and CoA. The enzyme has been purified 3,100-fold with a yield of 2.1% from 1.6 kg of rat liver using a purification scheme consisting of ammonium sulfate fractionation, ion exchange chromatography on CM (carboxymethyl)-cellulose and DEAE-cellulose, affinity chromatography on CoA-agarose, and gel filtration on Sephadex G-100. The final preparation gave a single band on polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme is 35,000 based on gel filtration, and sodium dodecyl sulfate-polyacrylamide electrophoresis in the presence and absence of 1% 2-mercaptoethanol. The enzyme was shown to be active on the D-isomer, but not on the L-isomer, of methylmalonyl-CoA by CD spectropolarimetry. The Km for D-methylmalonyl-CoA is 0.7 mM and the molar activity is 1,400 molecules of substrate/min/molecule of enzyme. At substrate concentrations of 0.5 mM, the relative rate of hydrolysis of CoA esters was as follows: D-methylmalonyl-CoA (100%), malonyl-CoA (16%), propionyl-CoA (3%), acetyl-CoA (1%), succinyl-CoA (less than 1%), and palmitoyl-CoA (less than 1%). This enzyme appears to account for the markedly increased amounts of methylmalonic acid that are excreted in the urine in cobalamin deficiency and in genetic disorders involving adenosylcobalamin or L-methylmalonyl-CoA mutase.