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Malate synthase from baker's yeast (5 kg) was purified 400–500‐fold to homogeneity. About 50–200 mg homogeneous enzyme were obtained within a week in a yield of 30% with reference to the total activity in cell‐free crude extracts. The enzyme, pl = 7.5, was pure as judged from ultracentrifugal and gel electrophoretic studies. Sedimentation and diffusion coefficients were determined: . The molecular weight of the synthase was found to be 175000 ± 10000 and 180000 ± 10000 by sedimentation/diffusion and by high‐speed sedimentation equilibrium respectively. It was concluded from these and other results that malate synthase has a molecular mass of 180000 ± 10000. The synthase on sodium dodecylsulfate gel electrophoresis was dissociated to yield a single specimen of Mr about 66000. The result indicates a composition of the native enzyme from three subunits of identical or nearly identical mass. The binding of acetyl‐coenzyme A to the synthase is independent of Mg2+ but that of glyoxylate is strictly dependent on the presence of Mg2+. Kinetic studies indicate that the malate synthase reaction follows a sequential random mechanism. The intermolecular isotopic effect, kH : k2H= 1.4, was determined with acetyl‐coenzyme A and [2H3]acetylcoenzyme A under several different experimental conditions and was shown to reflect different maximal velocities of the two substrates. An enzymic procedure for the preparation of doubly labelled [3H,14C]acetyl‐coenzyme A is also presented.